The immune responses of common carp, Cyprinus carpio L., injected with carp interleukin-1beta gene.

The function of the common carp, Cyprinus carpio L., interleukin-1beta (IL-1beta) gene was studied by DNA injection. To investigate the immune responses to IL-1beta, a plasmid construct of cytomegalovirus (CMV)-driven carp IL-1beta was injected into the epaxial muscle of carp. IL-1beta protein expressed in serum on 1, 3, and 5 days after plasmid injection was quantified by ELISA and immunohistochemistry. IL1-beta gene injection increased proliferation of the lymphocytes by phytohemagglutinin (PHA). Macrophage functions, such as production of superoxide anion and phagocytosis, also were stimulated by IL-1beta gene injection. Moreover, an increase in resistance to Aeromonas hydrophila infection was recorded in IL-1beta-injected fish compared with control fish. Thus, the cloned homolog of IL-1beta from carp has all the functional similarities to the mammalian IL-1beta gene.     

CXC chemokines and leukocyte chemotaxis in common carp (Cyprinus carpio L.)

CXC chemokines, structurally recognizable by the position of four conserved cysteine residues, are prominent mediators of chemotaxis. Here we report a novel carp CXC chemokine obtained through homology cloning and compare it with fish orthologues genes and with a second, recently elucidated, carp CXC chemokine. Phylogenetic analyses clearly show that neither CXC chemokine resembles any of the mammalian CXC chemokines in particular. However, basal expression is most prominent in immune organs like anterior kidney and spleen, suggesting involvement in the immune response. Furthermore we show that anterior kidney phagocyte-enriched leukocyte suspensions express both chemokines and that this expression is upregulated by brief (4 h) stimulation with PMA, but not lipopolysaccharide. Neutrophilic granulocyte-enriched leukocytes display chemotaxis to human recombinant CXCL8 (hrCXCL8; interleukin-8), confirming CXC chemokine mediated chemotaxis of neutrophilic granulocytes in teleost fish. Factors secreted from carp phagocytes are also capable of inducing chemotaxis and secretion of these factors into culture supernatants is upregulated by PMA. Finally we demonstrate involvement of both CXC chemokines as well as CXCR1 and CXCR2 in acute Argulus japonicus infection. Collectively the data presented implicate the involvement of CXC chemokines in chemotaxis of fish neutrophils in a fashion that shares characteristics with the mammalian situation. However, the CXC chemokines involved differ enough from those involved in neutrophil chemotaxis in mammals to warrant their own nomenclature.     

Adrenergic regulation of the innate immune response in common carp (Cyprinus carpio L.)

Catecholamines exert their physiological actions through α and β adrenergic receptors (ARs). As ARs are not exclusively expressed on neuroendocrine cells, but also on leukocytes, they may facilitate neuroendocrine modulation of immune responses. We sequenced the β_2a-AR in common carp, and studied its expression profile and involvement in the regulation of teleost innate immune responses.β_2a-AR messenger RNA was found to be constitutively expressed in brain areas, especially in the preoptic nucleus (NPO, homologous to the mammalian hypothalamus), and in immune organs. During the active phase of an in vivo inflammatory response, induced by i.p. zymosan treatment, β_2a-AR gene expression was up-regulated in the peritoneal leukocytes. Additionally, adrenaline in vitro reduced the synthesis of oxygen radical species and nitric oxide, while it enhanced arginase activity in fish phagocytes. Furthermore, in vitro adrenaline administration inhibited expression of pro-inflammatory cytokines, chemokines and their receptors. It is therefore hypothesized that adrenaline will down-regulate phagocyte skewing toward classical/innate polarization.     

The ontogeny of mucosal immune cells in common carp (Cyprinus carpio L.)

The ontogeny of carp (Cyprinus carpio L.) immune cells was studied in mucosal organs (intestine, gills and skin) using the monoclonal antibodies WCL38 (intraepithelial lymphocytes), WCL15 (monocytes/macrophages) and WCI12 (B cells). In addition, recombination activating gene 1 expression was examined in the intestine with real time quantitative PCR and in situ hybridization to investigate extrathymic generation of lymphocytes. WCL38⁺ intraepithelial lymphocytes (putative T cells) appeared in the intestine at 3 days post-fertilization (dpf), which is shortly after hatching but before feeding, implying an important function at early age. These lymphoid cells appear in the intestine before the observation of the first thymocytes at 3–4 dpf, and together with the expression of recombination activating gene 1 in the intestine, suggests that similar to mammals at least part of these cells are generated in the intestine. WCL15⁺ monocytes/macrophages appeared in the lamina propria of the intestine at 7 dpf, but considerably later in the epithelium, while WCI12⁺ (B) cells appeared in intestine and gills at 6–7 weeks. From these results it can be concluded that putative T cells occur much earlier than B cells, and that B cells appear much later in the mucosae than in other internal lymphoid organs (2 wpf).     

Organization of the NKEF gene and its expression in the common carp (Cyprinus carpio)

Natural killer enhancing factor (NKEF) is a member of the newly defined peroxiredoxin (Prx) family. Its functions are to enhance the cytotoxic capacity of natural killer cells and to prevent DNA and protein from being damaged by oxidative stress in the presence of thiol compounds. However, little is known about the structure and function of NKEF in lower vertebrates. We have recently cloned a cDNA encoding NKEF from the common carp (Cyprinus carpio) by use of suppression subtractive hybridization (SSH). In the present study, we used PCR to obtain a genomic DNA which covers the entire coding region of carp NKEF. In the 3363bp-long genomic sequence, six exons and five introns were identified. The carp NKEF gene has splice donor/acceptor site sequences at the boundaries of exons and introns, and contains two Val-Cys-Pro (VCP) motifs. The exon/intron organization of the carp NKEF gene shows complete conservation with other members of the Prx family. Genomic Southern blotting analyses suggest that carp has multiple copies of the NKEF gene. RT-PCR analyses reveal that carp NKEF has very different expression levels not only in tissues but also from individuals.     

Survival of heterophyid metacercaria in common carp (Cyprinus carpio)

Heterophyidae are small intestinal trematodes that infect vertebrates worldwide. Common carp (Cyprinus carpio) is one of the most preferred freshwater fish species by consumers in Asia, the region where fish-borne trematodes like Heterophyidae are most prevalent. How long Heterophyidae survive in common carp is unknown. The objective of this study was to quantify survival of Heterophyidae in common carp after experimental exposure. Fish of 0.18 g were either used as controls or exposed to 250 heterophyid cercaria for 24 h. Control fish did not become infected. Percentage infection of exposed fish at 0–2 (n?=?53), >2–10 (n?=?15), >10–20 (n?=?11), and >20–27 (n?=?33) weeks post exposure was 98, 80, 100, and 100 % respectively. The number of metacercaria per fish did not significantly decrease (P?=?0.19) during 27 weeks after exposure: exp [3.6200–0.0193?×?weeks post exposure]. All developed metacercaria were identified as Haplorchis spp. It was concluded that Heterophyidae may persist in carp for a long time, implying that harvestable carp are a risk to human health.     

Isolation and characterization of a novel CXC chemokine in common carp (Cyprinus carpio L.)

A novel CXC chemokine was identified for the first time in fish from common carp (Cyprinus carpio L.). The gene was obtained from the head kidney (HK) stimulated with LPS and Con A. The cDNA consists of 619 bp with a 37 bp 5' UTR and a 287 bp 3' UTR. An open reading frame of 368 bp encodes a 97 amino acid peptide, with a putative signal peptide of 20 aa. The gene has four cysteines residues, which are conserved, with first two cysteines separated with phenylalanine. By homology and phylogenetic analysis, the chemokine was found to be closer to human IP-10. Identities were significantly low to the CXC chemokines cloned from lamprey (Lampetra fluviatilis), flounder (Paralichthys olivaceus), rainbow trout (Onchorhynchus mykiss) and zebrafish (Danio rerio). The carp CXC chemokine contains three exons interrupted by two introns. The gene was transcribed from an early time point by stimulation with LPS and Con A. Organs in resting phase as well as stimulated expressed the gene.     

Perceptual brightness space in the carp (Cyprinus carpio L.)

An instrumental differentiation method was used to study the discrimination of the intensities of black/white (over the range 0.0082–0.214 W/m²; coordinates in the CIE-31 system: X=0.340, Y=0.354) and red (over the range 0.0035–0.106 W/m²; coordinates in the CIE-31 system: X=0.641, Y=0.342) stimuli in two carp fish (Cyprinus carpio L.). Confusion matrices were constructed using the probabilities of instrumental responses (bead seizing) on selection from a pair of stimuli (a conditioned stimulus and one of nine differentiating stimuli) in six series using a conditioned stimulus of defined brightness. Matrices of correlations between vectors (the stimuli used for the confusion matrices) were subjected to factor analysis to identify their intrinsic vectors. The perceptual brightness spaces for both black/white (two fish) and red (one fish) stimuli were found to be spherical in structure, resembling the major features of the human brightness space. The coordinate axes of this space were interpreted as representing excitation of two channels encoding intensity as brightness and darkness. M. V. Lomonosov Moscow State University. Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti, Vol. 45, No. 5, pp. 964–975, September–October, 1995.     

Cortisol induces apoptosis in activated B cells, not in other lymphoid cells of the common carp, Cyprinus carpio L.

In mammalian T and B cells glucocorticoids (GS) regulate development and selection through induction of apoptosis; more recently GS-induced apoptosis has also been implicated in the removal of circulating, activated T and B cells following an immune response. In an earlier report we have given the first evidence for cortisol-induced apoptosis as an immune regulator in an aquatic vertebrate, the common carp. Here we report on subpopulation-specific sensitivity of carp peripheral blood leukocytes (PBL) to cortisol-induced apoptosis. B cells, the most abundant leukocyte subpopulation in fish blood, are sensitised to cortisol-induced apoptosis by activation with the mitogens LPS or PHA. Cortisol-induced apoptosis in B cells is receptor mediated as it is blocked by the synthetic GS receptor blocker RU486. In contrast to what is known for mammalian lymphocytes, apoptosis in carp T cells is hardly affected by cortisol, both in unstimulated and in PHA-stimulated cell cultures. A culture supernatant of PHA-prestimulated PBL, containing IL-2-like activity, decreased spontaneous apoptosis in both T and B cells, but did not affect cortisol-induced apoptosis in B cells. Apoptosis in thrombocytes was unaffected by either mitogens, cortisol, or lymphocyte supernatant. The difference between mammalian and fish leukocyte sensitivity to cortisol is discussed in the light of differences in the immune response of mammals and fish.     

Isolation and characterisation of pentraxin-like serum proteins from the common carp Cyprinus carpio

There is increasing economic and ecological interest in the development of assays for the early detection of infection, disease activity and environmental stress in marine and freshwater animals. In humans the serum pentraxin C-reactive protein (CRP) is universally used as a clinical indicator of inflammation and underlying infection. As a first step towards assessing the potential of an immunoassay for CRP in fish, we have isolated and characterised common carp Cyprinus carpio CRP and a highly specific and sensitive anti-carp CRP polyclonal antibody has been raised. The results show levels of CRP in healthy fish similar to those found in healthy humans. A protein of unknown function, which displays the characteristic calcium-dependent phosphate monoester binding exhibited by CRP and some similarity to the known fish pentraxin sequences, has also been identified.     

Ultrastructural characterization of leucocytes in the pronephros of carp (Cyprinus carpio, L.)

In the pronephros of carp (Cyprinus carpio, L.) the following cells were found and ultrastructurally characterized: erythrocytes; lymphocytes and plasma cells; thrombocytes; neutrophilic, eosinophilic and basophilic granulocytes; phagocytic reticular cells and monocytes and non-phagocytic reticular cells. The cells could be separated on a Percoll continuous density gradient and relatively pure fractions could be obtained. In vitro phagocytosis of bacteria (Bacillus megaterium) was found in monocytes and neutrophilic granulocytes, while basophilic and eosinophilic granulocytes engulfed bacterial cells without actual endocytotic uptake.     

A chemokinetic factor in the carp Cyprinus carpio

The results describe experiments which demonstrate that the teleost Cyprinus carpio is capable of elaborating a substance which has analogous in vitro effects to that of mammalian chemokinetic lymphokines. In vitro stimulation of leucocytes from carp pronephros, both with antigens and mitogens, was used to provide culture supernatants which were applied to an in vitro chemokinetic assay. The chemokinetic factor-like activity could be detected in supernatants after one day of culture and at a dilution of 25%. The presence of lymphokines in fish supports the theory that lymphokine production is a fundamental property of vertebrate leucocytes and is of central importance in vivo cell-mediated immunity.     

Anti-idiotypic antibodies of IgM-type produced in carp (Cyprinus carpio L.).

Carp synthesize highly specific anti-idiotypic antibodies of the IgM class. Ant-idiotypic antibodies could be elicited in these animals by a human IgM myeloma protein and were detected in a passive hemagglutination assay. The agglutination was completely inhibited by approximately 0.15 microgram homologous antigen, whereas a 100 000-fold excess of a heterologous IgM myeloma protein of the same L chain type did not produce any inhibition. A possible subgroup specificity of carp anti-idiotypic antisera can be excluded.     

Localization of immune complexes and heat-aggregated immunoglobulin in the carp Cyprinus carpio L.

In carp Cyprinus carpio L. preimmunized with a protein antigen, human gamma globulin (HGG), rapid localization of antigen occurred in the splenic ellipsoids within 1 day of a secondary immunization given 8 weeks later. Furthermore, after injection of immune complexes or heat-aggregated HGG, antigen was trapped more quickly in the pronephros than when antigen was injected alone. These results support the conclusion that in fish, as in homoiotherms, this form of antigen trapping is antibody-dependent.     

Growth of organs and tissues in carp (Cyprinus carpio L.) larvae

Growth of some internal organs and tissues was studied in carp (Cyprinus carpio L.) in order to provide baseline information on the growth process during the first posthatching stages of fish, and to compare the trend observed in fish to that known in other animals. Growth of organs and tissues (liver, pancreas, trunk muscles, digestive tract, eyes, nervous system) was studied in rapidly growing carp (Cyprinus carpio L.) larvae by means of a quantitative histological method. Mean body weight of carp larvae rose from 1.8 mg at 2 days to 50.4 mg at 18 days. Between 4 and 18 days after hatching (yolk exhausted; exogenous food only), the average specific growth rates of organs were around 31% per day for the liver and pancreas, 28% per day for the trunk muscles, 26% per day for the digestive tract (without lumen) and 19% per day for the eyes and nervous system. Compared to the total volume of tissues, liver (k = 1.19), pancreas (k = 1.19), and trunk muscles (k = 1.09) showed positive allometry, digestive tract showed isometry (k = 1.00), and eyes (k = 0.76) and the nervous system (k = 0.73) showed negative allometry. This study demonstrated that the growth process of some organs (i.e. liver, pancreas and digestive tract) is different between larval and older stages of carp, and that the development of carp larvae exhibit the same order of allometry coefficients as do certain mammals (e.g. rabbit and lamb) in their early postnatal stages: nervous tissue, digestive tract, muscular tissue, liver.