Publications by authors named "Ilgiz Irnazarow"

28 Publications

  • Page 1 of 1

The many faces of transferrin: Does genotype modulate immune response?

Fish Shellfish Immunol 2020 Jul 11;102:511-518. Epub 2020 May 11.

Polish Academy of Sciences, Institute of Ichthyobiology and Aquaculture in Gołysz, Zaborze, 43-520, Chybie, Poland. Electronic address:

In this study, the expression of pro-inflammatory and iron metabolism genes were analysed under Trypanoplasma borreli (T. borreli) challenge in common carp. Three transferrin (Tf) genotypic groups: two homozygous - DD, GG, and heterozygous DG were intraperitoneally infected with a dose of 2.16 × 10/100 μL parasites. Organ and blood samples were collected at weekly intervals. During the infection period, mortality and parasitaemia were assessed along with measurements of blood iron concentrations and antibody levels. Expression of Tf, Fer, IRP1 and 2, TfR 1a and 1b, Hep, TNF α1 and α2, and IL-1 β was measured in the peak of parasitaemia and the week preceding the peak. Study revealed, that changes in iron blood level induced by parasite were not correlated with the activities of iron homeostasis genes. Neither iron content nor the specific antibody response correlated with survival. We demonstrate that challenged carp, display three distinct, Tf genotype dependent activity patterns of iron homeostasis genes expression. The expected, "classical" way of up-regulation represented homozygous DD individuals. In contrast, GG individuals demonstrated downward trend, while gene expressions of heterozygous DG carp could be defined as an intermediate. We speculate, whether this phenomenon is related to the transferrin molecule itself or to Tf-genotypes being markers of other factors, that influence the iron homeostasis genes activities. We discussed the role of alarmins in triggering the immune response. Distinct genes activating patterns of homozygous genotypes DD and GG had no consequences in terms of mortality rates caused by T.borreli. The highest mortality was observed in the heterozygous group DG. In conclusion, this study suggest that transferrin variant, but not iron blood concentration, has a significant impact on carp immune response to blood parasite infection. This research sheds a new light on the inflammation process and interaction between a host and invaders.
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http://dx.doi.org/10.1016/j.fsi.2020.05.001DOI Listing
July 2020

Anti-CyHV-3 Effect of Fluorescent, Tricyclic Derivative of Acyclovir 6-(4-MeOPh)-TACV .

J Vet Res 2019 Dec 24;63(4):513-518. Epub 2019 Oct 24.

Institute of Ichthyobiology and Aquaculture, Polish Academy of Sciences, 43-520 Chybie, Poland.

Introduction: (CyHV-3) is a virus infecting carp with disease symptoms of gill necrosis, fish discoloration, sunken eyes, and mortality reaching 90%. Several research groups have examined how to potentially abate the consequences of viral activity. Recently we showed that acyclovir inhibits CyHV-3 replication and in the present study we examined the anti-CyHV-3 activity of the tricyclic derivative of acyclovir 6-(4-MeOPh)-TACV (T-ACV), a fluorescent molecule known for higher lipophilicity than acyclovir, and therefore potentially better candidate for application .

Material And Methods: CCB and KF1 cell lines were incubated with T-ACV at concentrations of 0, 66.67, and 133.33 μM for three days and toxicity examined with MTT and CV assays. To investigate the antiviral activity of T-ACV, the lines were infected with CyHV-3 or mock infected and incubated for three days with the drug at concentrations of 0 or 66.67 μM. The activity of T-ACV was evaluated by plaque assay and TaqMan qPCR.

Results: T-ACV at a concentration of 66.67 μM displayed low toxicity and inhibited CyHV-3 activity by 13-29%, varying by cell line and method.

Conclusion: The low anti-CyHV-3 activity of T-ACV indicates that it would be reasonable to screen several tricyclic derivatives of acyclovir for such activity.
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http://dx.doi.org/10.2478/jvetres-2019-0065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6950444PMC
December 2019

Hormonal stimulation of carp is accompanied by changes in seminal plasma proteins associated with the immune and stress responses.

J Proteomics 2019 06 25;202:103369. Epub 2019 Apr 25.

Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland.

Hormonal stimulation in common carp is a routine practice to enhance sperm production and control gamete maturation. This study aimed to compare the proteome of carp seminal plasma between control and Ovopel-induced males using two-dimensional differential in-gel electrophoresis. Ovopel induction increased sperm volume, total sperm count, seminal plasma osmolality, and pH and decreased seminal plasma protein concentration. In total, 36 spots were identified (23 up- and 13 downregulated), corresponding to 23 proteins differentially abundant in seminal plasma after Ovopel induction (p < .05; fold change 1.2). The majority of proteins were associated with the immune and stress responses including the transport protein (hephaestin), antiproteases (fetuin, α2-macroglobulin, TIMP2), complement components (C3, complement factor B/C2A), regulator of the coagulation cascade (plasminogen), modulators of the innate immune response, such as intelectin, ApoA and ApoE, and the cathepsin/cystatin system, and stress response (enolase1). In addition, hormonal stimulation seems to be related to the proteins involved in lipid metabolism, signal transduction, and tissue remodeling. Our results suggest that hormonal stimulation is not just concomitant with the hydration of testis but also induces the synthesis and secretion of seminal plasma proteins involved in sperm maturation and protection against stress induced by administration of the exogenous hormone. SIGNIFICANCE: It is well known that hormonal stimulation of male fish induces the final maturation of spermatozoa. However, molecular and biochemical basis underlying hormone-induced changes in semen is unknown at present. This study for the first time reveals, using proteomic approach, that hormonal stimulation in addition to hydration of testis is accompanied by significant changes in seminal plasma proteins related mainly to immune and stress response, lipid metabolism, signal transduction and tissue remodeling. These changes are associated with gene expression and synthesis and secretion of seminal plasma proteins by reproductive tissues. Overall, our results provide a framework for understanding the molecular mechanism responsible for hormonal stimulation in the reproductive tract of fish males.
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http://dx.doi.org/10.1016/j.jprot.2019.04.019DOI Listing
June 2019

Influence of the Genetic Makeup of Common Carp on the Expression of Iron-related Genes During Infection.

J Vet Res 2018 Sep 10;62(3):285-290. Epub 2018 Dec 10.

Polish Academy of Sciences, Institute of Ichthyobiology and Aquaculture in Gołysz, Zaborze, 43-520 Chybie, Poland.

Introduction: Genes related to iron metabolism play an important role in inflammatory response. The objective of this study was to investigate the role of ferritin, transferrin receptors 1a and 1b, and transferrin genes in the response to blood parasite infection in common carp ( L.).

Material And Methods: Two genetically distinct carp groups were used: R3 carp, which are established as being sensitive to parasitic infection, and SA carp () of wild origin. An established challenge model with was applied. Challenged carp were sampled to determine their expression levels of transferrin receptors 1a and 1b, ferritin, and transferrin mRNA. Mortality and serum iron concentration were also measured.

Results: The study revealed contrasting differences in the expression profiles of all key iron regulatory genes except the transferrin gene. In the case of other parameters, significant differences were also observed.

Conclusion: Our results demonstrate that the level of parasitic infection depends on the blood iron status. This parameter was related to the origin of the fish.
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http://dx.doi.org/10.2478/jvetres-2018-0041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6295998PMC
September 2018

Acyclovir inhibits Cyprinid herpesvirus 3 multiplication in vitro.

J Fish Dis 2018 Nov 24;41(11):1709-1718. Epub 2018 Aug 24.

Institute of Ichthyobiology and Aquaculture, Polish Academy of Sciences, Chybie, Poland.

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is an aetiological agent of a virulent and lethal disease in common and koi carp. In this study, we examined in vitro the anti-CyHV-3 activity of acyclovir (ACV), nucleoside analogue commonly used against human herpesviruses, as well as acyclovir monophospate (ACV-MP). The cytotoxicity of the ACV and the ACV-MP for two common carp cell lines, CCB (Common carp brain) and KF1 (Koi carp fin 1), was determined by means of MTT and crystal violet assays. In subsequent studies, the concentration of 66.67 μM was applied. The ACV and the ACV-MP (66.67 μM) inhibited a cytopathic effect (CPE) induced by the CyHV-3 virus in the CCB (ACV by 66%, ACV-MP by 58%) and the KF1 (ACV by 25%, ACV-MP by 37%). The viral load measured by the means of TaqMan qPCR was reduced in a range of 67%-93% depending on the analogue, the cell line and the time of incubation. The expression of viral genes (ORF149, ORF3, ORF134 and ORF78) in CCB cells infected with the CyHV-3 was strongly downregulated within the range of 78%-91%. In summary, both the ACV and the ACV-MP can inhibit CyHV-3 replication in vitro.
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http://dx.doi.org/10.1111/jfd.12880DOI Listing
November 2018

Proteomic identification of seminal plasma proteins related to the freezability of carp semen.

J Proteomics 2017 06 24;162:52-61. Epub 2017 Apr 24.

Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland.

The variation in sperm freezability among individuals within a fish species is a major factor justifying the identification of useful predictive indicators of cryopreservation success. It is unknown at present whether the protein composition of fish seminal plasma affects sperm freezability. Therefore, the aims of this study were to compare the proteome of carp seminal plasma from semen rated as good (GF) and poor (PF) freezability by two-dimensional difference gel electrophoresis followed by MALDI-TOF/TOF mass spectrometry. The semen was classified as GF and PF based on sperm motility assessment after freeze/thawing. Five spots representing three proteins were more abundant in GF, while ten spots representing seven proteins were more abundant in PF seminal plasma. The majority of proteins present in higher abundance in PF seminal plasma were associated with the innate immune response. On the other hand, higher freezability was associated with proteins involved in the maintenance of sperm membrane integrity and antioxidative protection. These results indicate that carp semen freezability levels may be related to different seminal plasma protein profiles. Lower usefulness of spermatozoa in cryopreservation may be related to previous infection or stress leading to sublethal changes to sperm structure.

Significance: Sperm quality parameters such as motility, viability and sperm concentration have been used as predictive tools of sperm cryopreservation potential in fish species However, the usefulness of initial motility parameters as indicators of freezability varies among fish species and between individuals within a species. Recent studies in mammals revealed that male-to-male variability in cryoresistance can be attributed to differences in seminal plasma protein composition. To the best of our knowledge, no proteomic studies linking the protein composition of fish seminal plasma and freezing resilience have been performed in fish. Our results indicate for the first time that factors regulating how carp semen tolerate cryopreservation may be related to the different protein profiles of carp seminal plasma. The obtained results provide new insight into understanding the molecular mechanisms underlying cryoresistance of carp semen and provide a tool for the improvement of a long-term sperm preservation procedure.
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http://dx.doi.org/10.1016/j.jprot.2017.04.015DOI Listing
June 2017

Differential effects of alloherpesvirus CyHV-3 and rhabdovirus SVCV on apoptosis in fish cells.

Vet Microbiol 2015 Mar 23;176(1-2):19-31. Epub 2014 Dec 23.

Institute of Science and Technology in Medicine, School of Life Sciences, Keele University, ST5 5BG Keele, United Kingdom. Electronic address:

Whilst Herpesviridae, which infect higher vertebrates, actively influence host immune responses to ensure viral replication, it is mostly unknown if Alloherpesviridae, which infect lower vertebrates, possess similar abilities. An important antiviral response is clearance of infected cells via apoptosis, which in mammals influences the outcome of infection. Here, we utilise common carp infected with CyHV-3 to determine the effect on the expression of genes encoding apoptosis-related proteins (p53, Caspase 9, Apaf-1, IAP, iNOS) in the pronephros, spleen and gills. The influence of CyHV-3 on CCB cells was also studied and compared to SVCV (a rhabdovirus) which induces apoptosis in carp cell lines. Although CyHV-3 induced iNOS expression in vivo, significant induction of the genetic apoptosis pathway was only seen in the pronephros. In vitro CyHV-3 did not induce apoptosis or apoptosis-related expression whilst SVCV did stimulate apoptosis. This suggests that CyHV-3 possesses mechanisms similar to herpesviruses of higher vertebrates to inhibit the antiviral apoptotic process.
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http://dx.doi.org/10.1016/j.vetmic.2014.12.012DOI Listing
March 2015

Interaction between type I interferon and Cyprinid herpesvirus 3 in two genetic lines of common carp Cyprinus carpio.

Dis Aquat Organ 2014 Sep;111(2):107-18

Fish Disease Research Unit, Centre of Infectious Diseases, University of Veterinary Medicine Hannover, Bünteweg 17, 30559 Hannover, Germany.

Cyprinid herpesvirus 3 (CyHV-3) infection in common carp Cyprinus carpio L. and its ornamental koi varieties can induce the severe systemic disease known as koi herpesvirus disease. This disease is characterised by a rapid replication and spreading of the virus through multiple organs and results in a fast onset of mortality (starting on Day 6 post infection) in up to 100% of infected fish. During the first phase of viral infections, type I interferons (IFNs) have generally been proven to be essential in inducing an innate immune response; however, very little is known about the type I IFN response to herpesviruses in fish. The aim of this work was to study the type I IFN responses during CyHV-3 infection in 2 genetically divergent lines of common carp which presented differing survival rates. Our results show that CyHV-3 induced a systemic type I IFN response in carp, and the magnitude of type I IFN expression is correlated with the virus load found in skin and head kidney. In this in vivo experimental setup, the level of type I IFN response cannot be linked with higher survival of carp during CyHV-3 infection.
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http://dx.doi.org/10.3354/dao02773DOI Listing
September 2014

C-reactive protein and complement as acute phase reactants in common carp Cyprinus carpio during CyHV-3 infection.

Dis Aquat Organ 2014 Jul;109(3):187-99

Institute of Science and Technology in Medicine, School of Life Sciences, Keele University, ST5 5BG Keele, UK.

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a highly virulent and lethal disease of common carp Cyprinus carpio and its ornamental koi varieties. However, specific knowledge about immune mechanisms behind the infection process is very limited. We aimed to evaluate the effect of the CyHV-3 infection on the profile of 2 major components of the common carp immune acute phase response: the C-reactive protein (CRP) and the complement system. Common carp were infected with CyHV-3 by bath immersion. Fish were sampled before the infection and at 6, 12, 24, 72, 120 and 336 h post-infection for serum and head kidney, liver, gill and spleen tissues. CRP levels and complement activity were determined from the serum, whereas CRP- and complement-related genes (crp1, crp2, c1rs, bf/c2, c3, masp2) expression profiles were analysed in the tissues by quantitative PCR. Both CRP levels and complement activity increased significantly up to 10- and 3-fold, respectively, in the serum of infected fish during the challenge. Analysis revealed distinct organ- and time-dependent expression profile patterns for all selected genes. These results suggest that CRP and complement behave as acute phase reactants to CyHV-3 infection in common carp with an organ- and time-dependent response.
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http://dx.doi.org/10.3354/dao02727DOI Listing
July 2014

Isolation, characterisation and cDNA sequencing of a new form of parvalbumin from carp semen.

Reprod Fertil Dev 2014 Oct;26(8):1117-28

Department of Gamete and Embryo Biology, Semen Biology Group, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, 10-747 Olsztyn, Poland.

Parvalbumins (Pv) are calcium-binding proteins present mainly in the muscle and nervous system where they act as a Ca(2+) buffer. Our previous work demonstrated the presence of Pv-I in carp semen and indicated the presence of a second Pv (Pv-II). The purpose of the present work was to identify, purify and determine the full-length cDNA sequence of Pv-II from carp testis. Pv-II from seminal plasma was purified by ion-exchange chromatography (IEC) and preparative electrophoresis, while the Pv-II from spermatozoa was purified by IEC, gel filtration and preparative electrophoresis. The purified Pv-II was submitted to an analysis of molecular mass, isoelectric point (pI), amino-acid sequence and oligomerisation ability. The amino-acid sequence was used to construct primers and obtain the full-length cDNA sequence of seminal-specific Pv-II from carp testis. Analysis of the cDNA sequence indicated that carp-testis Pv-II was distinct from carp-muscle parvalbumins. Pv-II was distinct from Pv-I regarding sequence, molecular mass and pI. Both parvalbumins had the ability to form oligomers or to bind to other proteins. Carp seminal plasma had a protective effect against parvalbumin oligomerisation. Pv-II underwent post-translational modification such as n-acetylation and cysteinylation. The present study is the first to report the full-length cDNA sequence of parvalbumin from carp testis.
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http://dx.doi.org/10.1071/RD13181DOI Listing
October 2014

Biology and host response to Cyprinid herpesvirus 3 infection in common carp.

Dev Comp Immunol 2014 Apr 24;43(2):151-9. Epub 2013 Aug 24.

Aquatic Biotechnology Center of WCU Project, College of Veterinary Medicine, Gyeongsang National University, 900 Gajwa-dong, Jinju, Gyeongnam 660-710, South Korea; Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513, Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041, Japan.

Viruses from the family Alloherpesviridae form an aquatic clade of herpesviruses infecting fish and amphibia. Diseases caused by these herpesviruses are of increasing importance because of the high morbidity and mortality associated with the infection, and the difficulties in diagnosing latently infected carriers. Cyprinid herpesvirus 3 (CyHV-3) induces a severe disease and mortality in common carp and thus greatly affects carp aquaculture and trade. This review summarises advancements in the understanding of the infection process and the current knowledge on immune responses of carp to CyHV-3. A focus is laid on host genetics and immunity responsible for resistance/survival from the disease and on the viral mechanisms accountable for evasion of carp immune responses. As current knowledge of immune responses to CyHV-3 is still limited, perspectives for future studies are outlined. Analysing CyHV-3 fish-host interactions will be useful and thought-provoking for a basic understanding of fish immune responses.
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http://dx.doi.org/10.1016/j.dci.2013.08.015DOI Listing
April 2014

Dietary β-glucan stimulate complement and C-reactive protein acute phase responses in common carp (Cyprinus carpio) during an Aeromonas salmonicida infection.

Fish Shellfish Immunol 2013 Mar 2;34(3):819-31. Epub 2013 Jan 2.

Institute of Science and Technology in Medicine, School of Life Sciences, Keele University, ST5 5BG Keele, United Kingdom.

The effect of β-glucans as feed additive on the profile of C-reactive protein (CRP) and complement acute phase responses was studied in common carp Cyprinus carpio after exposition to a bacterial infection with Aeromonas salmonicida. Carp were orally administered with β-glucan (MacroGard®) for 14 days with a daily β-glucan intake of 6 mg per kg body weight. Fish were then intraperitoneally injected with either PBS or 1 × 10⁸ bacteria per fish and sampled at time 0, 6, 12, 24, 48, 72, 96 and 120 h post-injection (p.i.) for serum and head kidney, liver and mid-gut tissues. CRP levels and complement activity were determined in the serum samples whilst the gene expression profiles of CRP and complement related genes (crp1, crp2, c1r/s, bf/c2, c3 and masp2) were analysed in the tissues by quantitative PCR. Results obtained showed that oral administration of β-glucan for 14 days significantly increased serum CRP levels up to 2 fold and serum alternative complement activity (ACP) up to 35 fold. The bacterial infection on its own (i.e. not combined with a β-glucan feeding) did have significant effects on complement response whilst CRP was not detectably induced during the carp acute phase reaction. However, the combination of the infection and the β-glucan feeding did show significant effects on both CRP and complement profiles with higher serum CRP levels and serum ACP activity in the β-glucan fed fish than in the control fed fish. In addition, a distinct organ and time dependent expression profile pattern was detected for all the selected genes: a peak of gene expression first occurred in the head kidney tissue (6 h p.i. or 12 h p.i.), then an up-regulation in the liver several hours later (24 h p.i.) and finally up- or down-regulations in the mid-gut at 24 h p.i. and 72 h p.i. In conclusion, the results of this study suggest that MacroGard® stimulated CRP and complement responses to A. salmonicida infection in common carp.
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http://dx.doi.org/10.1016/j.fsi.2012.12.017DOI Listing
March 2013

Intestinal barrier of carp (Cyprinus carpio L.) during a cyprinid herpesvirus 3-infection: molecular identification and regulation of the mRNA expression of claudin encoding genes.

Fish Shellfish Immunol 2013 Jan 26;34(1):305-14. Epub 2012 Nov 26.

Fish Disease Research Unit, Centre of Infectious Diseases, University of Veterinary Medicine Hannover, Hannover, Germany.

As a major part of tight junctions in the intestinal epithelium of vertebrates, claudin proteins are crucial to develop a selective permeable function and to maintain integrity of the barrier. The intestine has been reported as one of the targeted tissue of the cyprinid herpesvirus 3 (CyHV-3) or koi herpesvirus (KHV) which causes major disease problems in carp production worldwide. To analyse the impact of the disease on the epithelial barrier of the intestine, carp claudin encoding genes were cloned, their tissue expression was described, and the abundance of gene transcripts in the intestine of carp under CyHV-3 infection was determined. Some of the carp claudin genes such as claudin-7 and -11 were expressed in various tissues, whilst others, like claudin-2 and -23, showed more tissue-specific expression profiles, which may reflect specific functions of these particular claudins. Along the gut axis, a spatial distribution of claudin gene expressions was found, with a lower abundance of gene transcripts in anterior regions of the intestine and increased expression in the distal section of the intestine, which might indicate specific functions of different regions in the intestinal tract of carp. In carp under CyHV-3 infection, an up-regulation in the expression of IFN-a2, IL-1beta and iNOS was observed, together with an elevation of transcriptional levels of claudin-2, -3c, -11, and -23. The data suggest that expression of claudins is involved in the reorganisation of the intestinal epithelium in CyHV-3-infected carp, which may be responsible for changes in the paracellular permeability. An increased expression of the claudins might be a response to the disturbance of the hydromineral balance in carp under CyHV-3 infection.
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http://dx.doi.org/10.1016/j.fsi.2012.11.010DOI Listing
January 2013

Cyprinid herpesvirus 3 infection disrupts the skin barrier of common carp (Cyprinus carpio L.).

Vet Microbiol 2013 Mar 7;162(2-4):456-470. Epub 2012 Nov 7.

Fish Disease Research Unit, Centre of Infectious Diseases, University of Veterinary Medicine Hanover, Bünteweg 17, D-30559 Hanover, Germany.

Cyprinid herpesvirus-3 (CyHV-3) is recognised as a pathogen which causes mass mortality in populations of carp, Cyprinus carpio. One of the characteristic symptoms of the disease associated with CyHV-3 infection is the occurrence of skin lesions, sloughing off the epithelium and a lack of mucus. Furthermore, fish then seem to be more susceptible to secondary infections by bacterial, parasitic or fungal pathogens which may cause further mortality within the population. The observed pathological alterations lead to the assumption that the carp skin barrier is strongly challenged during CyHV-3 associated disease. Therefore we examined mRNA expression of genes encoding inflammatory mediators, type I interferons, and the following skin defence molecules: antimicrobial peptides, claudins, and mucin. In addition, we monitored changes in the bacterial flora of the skin during disease conditions. Our results show that CyHV-3 associated disease in the skin of common carp leads to a reduction in mRNA expression of genes encoding several important components of the mucosal barrier, in particular mucin 5B, beta defensin 1 and 2, and the tight junction proteins claudin 23 and 30. This caused changes in the bacterial flora and the development of secondary bacterial infection among some individual fish. To our knowledge this is the first report showing that under disease conditions associated with virus infection, the mucosal barrier of fish skin is disrupted resulting in a higher susceptibility to secondary infections. The reported clinical signs of CyHV-3 skin infection can now be explained by our results at the molecular level, although the mechanism of a probable virus induced immunomodulation has to be investigated further.
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http://dx.doi.org/10.1016/j.vetmic.2012.10.033DOI Listing
March 2013

Interferon type I responses to virus infections in carp cells: In vitro studies on Cyprinid herpesvirus 3 and Rhabdovirus carpio infections.

Fish Shellfish Immunol 2012 Sep 7;33(3):482-93. Epub 2012 Jun 7.

Fish Disease Research Unit, Centre of Infectious Diseases, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany.

Interferons (IFNs) are secreted mediators that play a fundamental role in the innate immune response against viruses among all vertebrate classes. Common carp is a host for two highly contagious viruses: spring viraemia of carp virus (Rhabdovirus carpio, SVCV) and the Cyprinid herpesvirus 3 (CyHV-3), which belong to Rhabdoviridae and Alloherpesviridae families, respectively. Both viruses are responsible for significant losses in carp aquaculture. In this paper we studied the mRNA expression profiles of genes encoding for proteins promoting various functions during the interferon pathway, from pattern recognition receptors to antiviral genes, during in vitro viral infection. Furthermore, we investigated the impact of the interferon pathway (stimulated with poly I:C) on CyHV-3 replication and the speed of virus spreading in cell culture. The results showed that two carp viruses, CyHV-3 and SVCV induced fundamentally different type I IFN responses in CCB cells. SVCV induced a high response in all studied genes, whereas CyHV-3 seems to induce no response in CCB cells, but it induces a response in head kidney leukocytes. The lack of an IFN type I response to CyHV-3 could be an indicator of anti-IFN actions of the virus, however the nature of this mechanism has to be evaluated in future studies. Our results also suggest that an activation of type I IFN in CyHV-3 infected cells can limit the spread of the virus in cell culture. This would open the opportunity to treat the disease associated with CyHV-3 by an application of poly I:C in certain cases.
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http://dx.doi.org/10.1016/j.fsi.2012.05.031DOI Listing
September 2012

Reduced inflammatory response to Aeromonas salmonicida infection in common carp (Cyprinus carpio L.) fed with β-glucan supplements.

Fish Shellfish Immunol 2012 Jun 2;32(6):1051-7. Epub 2012 Mar 2.

School of Life Sciences, Keele University, Keele, Staffordshire, United Kingdom.

The objective of the present study was to determine the action of β-glucans as feed additives on the gene expression profile of some inflammatory-related cytokines from common carp (Cyprinus carpio L.) during the early stages of a non-lethal bacterial infection with Aeromonas salmonicida. β-glucan (MacroGard(®)), was administered daily to carp (6 mg per kg body weight) in the form of supplemented commercial food pellets for 14 days prior to infection. Control and treated fish were then intraperitoneally injected with PBS or 4×10(8) bacteria per fish and were sampled at time 0 and 6h, 12h, 1 day, 3 days and 5 days post-injection. Head kidney and gut were collected and the gene expression patterns for tnfα1, tnfα2, il1β, il6 and il10 were analyzed by quantitative PCR. Results obtained showed that treatment with β-glucans generally down-regulated the expression of all measured genes when compared to their corresponding controls. After injection, highest changes in the gene expression levels were obtained at 6h; particularly, in head kidney there was higher up-regulation of tnfa1 and tnfa2 in infected fish fed β-glucans in comparison to control feed; however, in gut there was a significant down-regulation of tnfα1, tnfα2, il1β and il6 in infected fish fed β-glucans. Analysis of carp specific antibodies against A. salmonicida 30 days after injection revealed their levels were reduced in the infected β-glucan group. In conclusion, a diet supplemented with β-glucan (MacroGard(®)) reduced the gene expression levels of some inflammation-related cytokines in common carp. Such a response appears to be dependent of organ studied and therefore the immunostimulant may be preventing an acute and potential dangerous response in gut, whilst enhancing the inflammatory response in head kidney when exposed to A. salmonicida.
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http://dx.doi.org/10.1016/j.fsi.2012.02.028DOI Listing
June 2012

Gene expression analysis of common carp (Cyprinus carpio L.) lines during Cyprinid herpesvirus 3 infection yields insights into differential immune responses.

Dev Comp Immunol 2012 May 24;37(1):65-76. Epub 2011 Dec 24.

Polish Academy of Sciences, Institute of Ichthyobiology & Aquaculture in Gołysz, Kalinowa 2, 43-520 Chybie, Poland.

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is the etiological agent of a virulent and lethal disease in common and koi carp. This study aimed to determine the genetic basis underlying the common carp immune response to the CyHV-3 virus. Two common carp lines (R3 and K) were infected with CyHV-3 by immersion. The R3 line presented a 20% higher survival rate compared to the K line and significantly lower viral loads as measured at day 3 post infection (p.i.). Microarray analysis using a common carp slides containing a number of 10,822 60-mer probes, revealed that 581 genes in line K (330 up-regulated, 251 down-regulated) and 107 genes in line R3 (77 up-regulated, 30 down-regulated), showed at least a 2-fold difference in expression at day 3 p.i. compared to day 0. Genes which showed at least a 4-fold difference in expression in both lines were selected as potential markers of a CyHV-3 infection in common carp. Additionally, 76 genes showed at least 2-fold differentially expression between K and R3 lines at day 3 p.i. Significantly higher expression of several immune-related genes including number of those which are involve in pathogen recognition, complement activation, MHC class I-restricted antigen presentation and development of adaptive mucosal immunity was noted in more resistant R3 line. Further real-time PCR based analysis provided evidence for higher activation of CD8(+) T cells in R3 line. This study uncovered wide array of immune-related genes involved into antiviral response of common carp toward CyHV-3. It is also demonstrated that the outcome of this severe disease in large extent could be controlled by genetic factors of the host.
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http://dx.doi.org/10.1016/j.dci.2011.12.006DOI Listing
May 2012

Isolation and characterization of transferrin from common carp (Cyprinus carpio L) seminal plasma.

Fish Shellfish Immunol 2010 Jul 26;29(1):66-74. Epub 2010 Feb 26.

Department of Gamete and Embryo Biology, Semen Biology Group, Institute of Animal Reproduction and Food Research, 10-747 Olsztyn, Poland.

Transferrin (Tf) in fish is recognized as a component of non-specific humoral defense mechanisms against bacteria. It is a major protein of common carp seminal plasma but its structure and localization in carp testis is unknown. In this study we developed a simple and efficient three-step purification procedure consisting of affinity chromatography (Con A-Sepharose), hydrophobic interaction chromatography (Phenyl Sepharose) and gel filtration (Superdex 200). The molecular mass of Tf has been determined to be 73.6 kDa and isoelectric point 5.1. The peculiar characteristics of carp transferrin were the lack of carbohydrate component and binding of iron ions by only one functional iron-binding site. Western blot analysis revealed a strong similarity of carp seminal plasma Tf to carp blood Tf and Tf from seminal plasma of other cyprinids but a lower similarity to salmonid and percid fishes. Tf was localized to the blood vessels of the carp testis which strongly suggest that most Tf of carp seminal plasma originates from blood. In conclusion, seminal plasma Tf has a unique structure and is similar or identical to blood Tf.
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http://dx.doi.org/10.1016/j.fsi.2010.02.015DOI Listing
July 2010

Resistance of common carp (Cyprinus carpio L.) to Cyprinid herpesvirus-3 is influenced by major histocompatibility (MH) class II B gene polymorphism.

Fish Shellfish Immunol 2009 May 27;26(5):737-43. Epub 2009 Mar 27.

Polish Academy of Sciences, Institute of Ichthyobiology & Aquaculture in Gołysz, Zaborze, Chybie, Poland.

The role of MH class II B (Cyca-DAB1-like) genes in resistance of common carp (Cyprinus carpio L.) to Cyprinid herpesvirus-3 (CyHV-3), also known as koi herpesvirus (KHV) was analysed. The material consisted of 934 fish from six carp crosses. Fish were challenged with CyHV-3 at an age of 7 and 10 months. During challenge experiments the peak of mortality caused by CyHV-3 was observed at days 8-12 p.i. and the overall cumulative mortality reached 79.9%. Among six Cyca-DAB1-like genotypes, revealed by PCR-RF-SSCP analysis, one genotype (E) was found associated with higher resistance to CyHV-3. Three other genotypes (B, H and J) could be linked to higher susceptibility to CyHV-3. 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. Our data indicate that MH class II B genes could be used as potential genetic markers in breeding of common carp for resistance to this virus.
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http://dx.doi.org/10.1016/j.fsi.2009.03.001DOI Listing
May 2009

The induction of nitric oxide response of carp macrophages by transferrin is influenced by the allelic diversity of the molecule.

Fish Shellfish Immunol 2009 Apr 19;26(4):632-8. Epub 2008 Oct 19.

Polish Academy of Sciences, Institute of Ichthyobiology and Aquaculture, Gołysz, 43-520 Chybie, Poland.

The central role of transferrin (Tf) as an iron transporting protein has been extended by observations that modified versions of Tf also participate in the regulation of innate immunity. We report on the isolation of two carp Tf proteins (alleles D and G) to purity using rivanol precipitation and ion-exchange chromatography, and describe the activation of head kidney-derived carp macrophages by cleaved Tf. We demonstrate the superiority of the D-type over the G-type Tf in inducing nitric oxide (NO) and confirm previous observations that full-length Tf cannot induce NO in fish macrophages. We believe that cleaved Tf fragments should be considered to be "alarmins". We discuss the possibility that parasites such as Trypanoplasma borreli cleave Tf and use Tf fragments to their advantage by modulating the NO induction in carp macrophages.
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http://dx.doi.org/10.1016/j.fsi.2008.10.007DOI Listing
April 2009

Genetic resistance of carp (Cyprinus carpio L.) to Trypanoplasma borreli: influence of transferrin polymorphisms.

Vet Immunol Immunopathol 2009 Jan 19;127(1-2):19-25. Epub 2008 Sep 19.

Polish Academy of Sciences, Institute of Ichthyobiology and Aquaculture, Chybie, Poland.

In serum most of the iron molecules are bound to transferrin (Tf), which is a highly polymorphic protein in fish. Tf is an essential growth factor for mammalian trypanosomes. We performed a series of experiments with Trypanoplasma borreli to detect putative correlations between different Tf genotypes of common carp (Cyprinus carpio L.) and susceptibility to this blood parasite. Five genetically different, commercially exploited carp lines (Israelian 'D', Polish 'R2' and 'K', Ukrainian 'Ur', Hungarian 'R0') and a reference laboratory cross ('R3xR8') were challenged with T. borreli and parasitaemia measured to determine susceptibility to the parasite. Among the commercial carp lines, Israelian 'D' carp were identified as most and Polish 'R2' carp as least susceptible, and used to produce a next generation and reciprocal crosses. These progenies were challenged with T. borreli and parasitaemia measured. We demonstrated significant effects of genetic background of the carp lines on susceptibility to T. borreli. This genetic effect was preserved in a next generation. We also observed a significant male effect on susceptibility to T. borreli in the reciprocal crosses. Serum samples from a representative number of fish from two infection experiments were used for Tf genotyping by polyacrylamide gel electrophoresis (PAGE), identifying DD, DG and DF as most frequent Tf genotypes. We could detect a significant association of the homozygous DD genotype with low parasitaemia in the least susceptible 'R2' (and 'K') carp lines and the lack of a such an association in the most susceptible 'D' carp line. Upon examination of parasite growth in vitro in culture media supplemented with 3% serum taken from fish with different Tf genotypes, we could show a faster decrease in number of parasites in culture media with serum from DD-typed animals.
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http://dx.doi.org/10.1016/j.vetimm.2008.09.006DOI Listing
January 2009

Allelic discrimination, three-dimensional analysis and gene expression of multiple transferrin alleles of common carp (Cyprinus carpio L.).

Fish Shellfish Immunol 2009 Apr 12;26(4):573-81. Epub 2008 Sep 12.

Institute of Ichthyobiology and Aquaculture, Polish Academy of Sciences, Gołysz, 43-520 Chybie, Poland.

We cloned and sequenced four different transferrin (Tf) alleles (C, D, F and G) of European common carp (Cyprinus carpio carpio L.) and studied allelic diversity with respect to differences in sequence, constitutive transcription and three-dimensional structure. Most of the disulfide bonds were conserved between human and carp Tf, and modeling confirmed the overall conservation of the three-dimensional structure of carp Tf. While the iron-binding sites in the C-lobe of carp Tf were completely conserved, in the N-lobe the majority of iron-coordinating residues were not conserved. This may have a serious impact on the ability of carp Tf to bind iron with both the C- and N-lobe. In contrast to human Tf, we could not detect potential N-glycosylation sites in carp Tf, which does not seem to be a glycoprotein. Comparison of the cDNA of the four Tf alleles of carp indicated 21 polymorphic sites of which 13 resulted in non-synonymous changes. Allelic diversity did not seem to influence the overall conservation of carp Tf. Neither the iron binding sites nor the receptor binding of carp Tf seemed influenced by allelic diversity. Possibly, interaction with pathogen-associated receptors for Tf could be influenced by allelic diversity. Basal gene expression of Tf alleles D and G was especially high in carp liver. Although we could detect a higher transcription level of allele D than of Tf allele G in head kidney, thymus and spleen, the differences seem minor with respect to the very high transcription level in liver. Preliminary results with Tf-typed serum suggest a difference in the ability of Tf alleles D and G to modulate LPS-induced NO production in carp macrophages.
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http://dx.doi.org/10.1016/j.fsi.2008.08.017DOI Listing
April 2009

Classical crosses of common carp (Cyprinus carpio L.) show co-segregation of antibody response with major histocompatibility class II B genes.

Fish Shellfish Immunol 2009 Mar 6;26(3):352-8. Epub 2008 Sep 6.

Polish Academy of Sciences, Institute of Ichthyobiology and Aquaculture in Gołysz, Zaborze, ul. Kalinowa 2, 43-520 Chybie, Poland.

In cyprinids, two paralogous groups of major histocompatibility (MH) class II B genes, DAB1 and DAB3, have been reported but have not been studied in detail. In our study on MH association with immune responsiveness in common carp (Cyprinus carpio L.) we have taken a long-term approach using divergent selection for antibody production. We report the co-segregation of Cyca-DAB1-like and Cyca-DAB3-like genes with antibody response, in backcrosses to high- and low-responsive parental carp lines. We show that the presence of Cyca-DAB1-like, but not Cyca-DAB3-like genes, preferentially leads to a high DNP-specific antibody response in carp. Background genes other than Cyca-DAB genes also influenced the level of antibody response. Our data support the hypothesis of a genetic control by Cyca-DAB genes on the antibody response measured. We could not detect an association of the Cyca-DAB genes with disease resistance to the parasite Trypanoplasma borreli. Sequence information, constitutive transcription levels and our co-segregation data indicate that both paralogous Cyca-DAB1-like and Cyca-DAB3-like groups represent functional MH class II B genes. Previously defined differences in allelic diversity between Cyca-DAB1-like genes, especially, identify Cyca-DAB1 as the most interesting DAB gene for further study in common carp.
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http://dx.doi.org/10.1016/j.fsi.2008.08.011DOI Listing
March 2009

Serum CRP-like protein profile in common carp Cyprinus carpio challenged with Aeromonas hydrophila and Escherichia coli lipopolysaccharide.

Dev Comp Immunol 2008 13;32(11):1281-9. Epub 2008 May 13.

School of Life Sciences, Huxley Building, Keele University, Staffordshire ST5 5BG, UK.

The potential of C-reactive protein (CRP)-like proteins to be used as a biomarker of health status in cultured carp obtained from various European fish lines has been assessed. Varying CRP-like protein levels in the serum of carp were monitored using an indirect competitive enzyme-linked immunosorbent assay. CRP-like protein basal levels in normal fish varied between carp lines, ranging on average from 2.9+/-0.15 to 12.57+/-1.19 microg ml(-1). Serum levels of CRP-like protein in carp were observed to increase several fold in fish infected with the pathogen Aeromonas hydrophila. However, carp injected with Escherichia coli lipopolysaccharide (LPS) serotype 0111:B4 did not exhibit an increase in CRP-like proteins levels.
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http://dx.doi.org/10.1016/j.dci.2008.04.004DOI Listing
October 2008

Application of PCR-RF-SSCP to study major histocompatibility class II B polymorphism in common carp (Cyprinus carpio L.).

Fish Shellfish Immunol 2008 Jun 5;24(6):734-44. Epub 2007 Dec 5.

Polish Academy of Sciences, Institute of Ichthyobiology and Aquaculture in Golysz, Zaborze, ul. Kalinowa 2, Chybie, Poland.

A variety of methods have been applied for the characterization of major histocompatibility (MH) polymorphism in fish. We optimized a technique designated polymerase chain reaction-restriction fragments-single strand conformation polymorphism (PCR-RF-SSCP) for screening a large number of individuals for the Cyca-DAB1 and Cyca-DAB2 genes polymorphism in common carp. The advantages of this technique are simplicity, high sensitivity and low costs. PCR-RF-SSCP analysis revealed different genotypes consisting of unique combinations of the Cyca-DAB1 and Cyca-DAB2 sequences with the number of SSCP bands clearly correlating with the degree of heterozygosity of the Cyca-DAB1 and Cyca-DAB2 genes. We found four alleles for Cyca-DAB1 (*02-*05) gene but only one allele for Cyca-DAB2 (*02) and noted that the Cyca-DAB2 gene was either homozygous or absent. PCR-RF-SSCP analysis of n=79 carp individuals challenged with Aeromonas hydrophila indicated that individuals bearing no Cyca-DAB2 gene showed higher cumulative mortality and lower bacterial agglutination titers during the experiment. We suggest that our PCR-RF-SSCP method can be used to study correlations of different MH class II B genotypes/alleles with resistance of common carp to specific pathogens.
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http://dx.doi.org/10.1016/j.fsi.2007.11.015DOI Listing
June 2008

Polymorphism of transferrin of carp seminal plasma: relationship to blood transferrin and sperm motility characteristics.

Comp Biochem Physiol B Biochem Mol Biol 2007 Dec 1;148(4):426-31. Epub 2007 Aug 1.

Semen Biology Group, Institute of Animal Reproduction and Food Research, 10-747 Olsztyn, ul. Tuwima 10, Poland.

Transferrin (Tf) is a major protein of carp (Cyprinus carpio) seminal plasma. Its relationship with milt quality is unknown. In this study, we sought to determine if Tf is polymorphic in carp seminal plasma and if this polymorphism is related to sperm motility characteristics. We screened males of purebred common carp line (Polish line R6) for Tf polymorphism in blood plasma. The majority of Tf genotypes represented only DD and DG variants. We then collected milt from preselected DD and DG genotypes and tested their sperm motility characteristics using computer-aided sperm analysis (CASA). Tf polymorphism in seminal plasma was found to be identical with that of blood. However, the relationships between Tf polymorphism and iron metabolic parameters were different for blood and semen. These data suggest different regulation of Tf in liver and testis. We found substantial differences in sperm motility characteristics between both genotypes. Spermatozoa of DG males were characterized by lower curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), higher linearity (LIN) and straightness (STR) of movement as compared to DD males. No differences were found in other sperm characteristics such as sperm concentration and percentage of sperm motility. Our results suggest that sperm motility parameters are related to Tf polymorphism and therefore this polymorphism may be related to sperm competitive ability.
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http://dx.doi.org/10.1016/j.cbpb.2007.07.011DOI Listing
December 2007

Differential transcription of multiple forms of alpha-2-macroglobulin in carp (Cyprinus carpio) infected with parasites.

Dev Comp Immunol 2008 26;32(4):339-47. Epub 2007 Jul 26.

Polish Academy of Sciences, Institute of Ichthyobiology and Aquaculture, Gołysz, 43-520 Chybie, Poland.

Alpha-2-macroglobulin (a2M) is a non-specific protease inhibitor involved in host defense mechanisms, inhibiting both endogenous and exogenous proteases. It is unique among the plasma anti-proteases with respect to the diversity of proteases that it can inactivate. Carp a2M consists of an alpha and beta chain of which the first includes the bioactive regions. Previously, three a2M alpha chain sequences were reported for East-Asian common carp. We studied a2M alpha chain variability in European common carp and report the cloning of a fourth a2M alpha chain with distinct sequence diversity in the bait region. The role of a2M in the immune response to parasites was studied in the liver of carp infected with Trypanoplasma borreli or with Ichthyophthirius multifiliis. Quantitative gene transcription analysis showed a differential regulation of the four isoforms, most clearly seen in infections with I. multifiliis. A2M3 was the only a2M isoform with a highly upregulated transcription during infection, suggesting that this particular isoform is of foremost biological importance.
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http://dx.doi.org/10.1016/j.dci.2007.06.007DOI Listing
May 2008

Genetic differences in natural antibody levels in common carp (Cyprinus carpio L.).

Fish Shellfish Immunol 2006 Oct 20;21(4):404-13. Epub 2006 Mar 20.

Polish Academy of Sciences, Institute of Ichthyobiology and Aquaculture, Gołysz, 43-520 Chybie, Poland.

In mammals, natural antibodies (Nabs) are mostly of the IgM isotype and can bind to a particular antigen or pathogen even if the host has never been exposed. Despite their early detection and abundance, the exact role and genetic control of Nabs remain unclear. We have used an indirect ELISA with three different antigens (keyhole limpet haemocyanin, chicken ovalbumin and bovine serum albumin) to demonstrate the ubiquitous presence of Nabs in common carp. Serum levels of Nabs increased with age, i.e. 10-month-old fish showed higher levels than 4-month-old fish. Also, fish grown in earth ponds showed higher levels of Nabs than fish grown in a clean environment of UV-treated water. Furthermore, we show that Nabs are present in different levels in the serum of carp lines with a different genetic background, suggestive of a genetic control. These genetic differences were independent of antigen, age and environment. Genetic differences in levels of Nabs could not unequivocally be related to differences in survival under farmed conditions. The possibilities for using levels of Nabs as marker criterion for selection for genetic disease resistance are discussed.
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http://dx.doi.org/10.1016/j.fsi.2006.01.005DOI Listing
October 2006