Healthy Carp Research Articles

Humoral immune factors modulated by copper and chitosan in healthy or parasitised carp (Cyprinus carpio L.) by Ptychobothrium sp. (Cestoda)

As an environmental protection point of view, the potential toxicity of chitosan on aquatic animal health, alone or associated with copper must be investigated. Fish possess defence mechanisms to counteract the impact of toxics. The non-cellular and non-specific immune defences (total immunoglobulin, ceruloplasmin, lysozyme and potential killing activity of phagocytic cells) can be modulated by the potential environmental pollutants but also by natural stimulants such as bacteria, viruses or parasites. In this study, we investigate the potential toxicity of copper (0.1 and 0.25 mg/L) or chitosan (75 and 150 mg/L) and the combination copper and chitosan (0.1 and 75 mg/L, respectively) on two groups of carp: healthy or parasitised by Ptychobothrium sp. Fish exposed to water-soluble chitosan for 96 h had significantly high levels of natural antibodies in plasma. Moreover, activities of lysozyme and ceruloplasmin were also increased in plasma after the same treatment. The exposition of fish to copper have shown apparently contradictory effects on the immune parameters measured but, significant increase of this bacteriolytic activity was observed, particularly in head kidney after 4 days of treatment of fish with copper. The two products may induce separately an acute, short and local inflammatory acute phase response by stimulating some components of the innate immune response of healthy fish. The mixture seems to reduce the impact of the each product due to the physical and chemical properties of chitosan to complex with copper. The responses of humoral immune factors of treated carp was modulated by the presence of the parasite, as shown by the high elevation of lysozyme activity observed in parasitised carps after exposition to copper and by increases in natural antibodies levels observed in parasitised carp treated with the copper–chitosan mixture. This could indicate an additive effect on the stress response mediated by parasite. It occurred a greater stress response in the parasitised group than healthy group exposed to the same treatment evoking an additive effect. So, it is important to specify the health status of organisms to understand responses of immunological markers in fish.

First Detection of Koi Herpesvirus in Cultured Common Carp Cyprinus carpio in Japan

ABSTRACT-Mass mortality among common carp Cyprinus carpio, especially in large-sized fish cultured in Lake Kasumigaura, has occurred since early October 2003. The affected fish swam lethargically near the surface. A histopathological examination revealed hyperplasia and necrosis of the epithelial cells in the gills. DNA of the koi herpesvirus (KHV) was detected in the gills and kidneys of moribund fish by polymerase chain reaction (PCR) assay. The virus could not be isolated from the tissues, but the inoculation of gill homogenate from affected fish into healthy carp reproduced the disease conditions and killed the fish. KHV was detected from the gills of the experimentally infected fish by PCR, and the virus was isolated from those fish with KF-1 cells.

Molecular cloning and characterization of crucian carp ( Carassius auratus L.) interferon regulatory factor 7

Interferon (IFN) can induce an antiviral state via interferon-regulatory transcription factors (IRFs), which bind to and control genes directed by the interferon-stimulated response element (ISRE). Here we describe a fish IRF, termed CaIRF7, cloned from a subtractive cDNA library which is constructed with mRNAs obtained from crucian carp ( Carassius auratus L.) blastulae embryonic (CAB) cells infected by UV-inactivated GCHV and mock-infected cells. CaIRF7 cDNA was found to be 1816 bp in length, with a 42 bp 5′UTR and a 508 bp 3′UTR. The open reading frame translates into 421 amino acids in which a DNA-binding domain (DBD) containing the repeated tryptophan motif and IRFs association domain have been identified. Like chicken GgIRF3, CaIRF7 was most similar to mammalian IRF7 with 27 to 30% identity overall and some 37% identity in their DBDs. A single transcript of 1.9 kb was detected in virally induced CAB cells by virtual Northern blotting. RT–PCR analysis revealed a wide tissue distribution of CaIRF7 constitutive expression, with detectable transcript in non-infected CAB cells and various tissues of healthy crucian carp. In addition, CaIRF7 expression was differentially increased by stimulation of the CAB cells with active GCHV, UV-inactivated GCHV or CAB IFN, indicating that the activation of CaIRF7 was directly regulated by IFN.

Differential expression and haplotypic variation of two interleukin-1beta genes in the common carp (Cyprinus carpio L.).

Interleukin-1beta (IL-1beta) is a central component in innate immunity and the inflammatory response of mammals. Only recently, the first non-mammalian IL-1beta sequences were published. In this study, we describe a second IL-1beta sequence (IL-1beta2) in carp with 74% amino acid identity to the carp IL-1beta1 sequence. The existence of two IL-1beta copies in the carp genome probably originates from the tetraploid nature of the species. In contrast to the first carp IL-1beta sequence, IL-1beta2 is represented by multiple genes with 95-99% identity. Detection of several IL-1beta2 sequences within individual homozygous fish suggests the presence of multiple copies of the IL-1beta2 gene in the carp genome, possibly as a result of subsequent gene duplication of IL-1beta2. In vivo, constitutive mRNA expression of both IL-1beta genes was found in healthy carp. IL-1beta2 mRNA expression could be up-regulated in head kidney cells similar to carp IL-1beta1, in vivo by infection with Trypanoplasma borreli and in vitro by stimulation with lipopolysaccharide (LPS). Cortisol, the major glucocorticoid in fish, is an endocrine-derived fator mediating IL-1beta expression. Although constitutive IL-1beta expression was inhibited by a physiological dose of cortisol, cortisol synergistically enhanced LPS-induced IL-1beta expression in carp. Involvement of the transcription factor nuclear factor (NF)-kappaB in expression of IL-1beta1 and IL-1beta2 was demonstrated. Ratio of IL-1beta expression was determined and this showed IL-1beta1 mRNA expression to be at least tenfold higher compared with IL-1beta2. The possibilities of IL-1beta2 being a functional gene or approaching pseudogene status are discussed.

The haemoflagellate Trypanoplasma borreli induces the production of nitric oxide, which is associated with modulation of carp (Cyprinus carpio L.) leucocyte functions

In an attempt to characterise the role of nitric oxide (NO) in immune responses of carp, carp leucocytes obtained during an acute T. borreli infection were examined, for their capacity to generate NO. In a second set of experiments the impact NO on viability of the parasite and on the modulation of functional carp leucocyte responses were testedin vitro . Both in carp head-kidneys and in the peripheral blood, the fractions of lymphoblasts among separated leucocytes were increased. However, the relative proportions of granulocytes among head-kidney leucocytes (HKL) significantly decreased during infection, whereas granulocytes appeared among peripheral blood leucocytes (PBL). The cellular dynamics of HKL and PBL of infected carp were paralleled by an enhanced spontaneous NO release in vitro. NO production was further increased after addition of viable parasites to these cultures. The hypothesis that NO had a possible role in granulocyte activation and lymphocyte proliferation in carp was supported by the reduction of mitogen-induced proliferative responses of PBL from healthy carp in the presence of NO donor substances. The negative effects of NO on lymphocyte proliferation were contrasted by enhancing effects on granulocyte functions: the inhibition of NO generation in T. borreli -stimulated HKL cultures by the l-arginine analogue L-NMMA reduced the viability of granulocytes and their phagocytic activity. Even massive amounts of nitric oxide produced by donor substances (up to 600μmol l−1 NO−2) caused no reduction in the numbers of viable T. borreli flagellates in vitro. Thus, in carp, T. borreli seems to induce high amounts of NO in vivo which are apparently not harmful for the parasite but which may interfere with co-ordinated interactions of activated cells aiming at the defence of the parasite.

Antioxidant response modulated by copper in healthy or parasitized carp ( Cyprinus carpio L.) by Ptychobothrium sp. (Cestoda)

An increased antioxidant response (catalase, glutathione S-transferase (GST) and glutathione reductase (GRd) activities in liver and GST activity in head kidney) was observed in carp parasitized by Ptychobothrium sp. compared to healthy fish. In case of a copper contamination of these fish, the decrease in enzymatic activities observed was less pronounced in parasitized than in healthy carp.

The effect of dietary immunomodulation upon Edwardsiella tarda vaccination in healthy and immunocompromised Indian major carp (Labeo rohita)

In order to determine the impact on disease resistance of four dietary immunomodulators viz., β-1,3 glucan, levamisole, vitamins C and E, in an important farmed Indian major carp species, rohu (Labeo rohita Ham.), fish were fed diets containing various levels of these substances during a 60 day trial. Aflatoxin B1(AFB1) at 1·25mg kg−1body weight was injected intraperitoneally (i.p.) into fish to induce an immunosuppressive state on the first day of the experiment in some individuals. The fish were vaccinated against formalin-killedEdwardsiella tarda vaccine on day 30 of the experiment. Specific immunity, as measured by bacterial agglutination titre and disease resistance against E. tarda, was determined at the end of the trial. The results demonstrate that all the four immunomodulators were capable of significantly (P< 0·05) increasing specific immunity and reducing mortality in immunocompromised fish but failed to enhance specific immunity and protection in healthy fish. The increased bacterial agglutination titre by β-1,3 glucan, and reduced mortality losses by both β-1,3 glucan and levamisole were marked in healthy vaccinated fish compared with their controls. Similarly, all four substances significantly reduced the mortality rates in immunocompromised and healthy unvaccinated fish. Out of these four substances, glucan was recorded to be the most effective immunomodulator in rohu. The present results suggest that the introduction of these substances into the diet of fish grown in farms under immunosuppressive/stressful conditions could increase their resistance to infection by reducing mortality rates and offer economic benefits.

Qualitative and quantitative cytometric analysis of peripheral blood leukocytes in carps (Cyprinus carpio)

The studies were performed involving qualitative and quantitative cytometric analysis of peripheral blood leukocytes in healthy carps at various stages of their ontogeny, i.e., in 3- to 29-month old carps. Three populations of leukocyte line cells were distinguished, which differed in forward scatter (FSC) and side scatter (90o, SSC) of laser light. The most abundant leukocyte pool was present in the eldest (23- to 29-month old) fish. Lower numbers of the cells were observed in the youngest (3- to 9-month old) carps while the lowest levels of the cells were detected in carps of the moderate age (11- to 21-month old). The leukocyte populations, distinguished on grounds of FSC/SSC characteristics, were suggested to correspond to lymphocytes/thrombocytes (low FSC, low SSC), granulocytes (high FSC, high SSC) and monocytes (high FSC, low SSC).

Pathogenesis of Iridovirus: in vitro Influence on Macrophage Activity and Cytokine-Like Protein Production in Fish

Pathogenesis of Iridovirus: in vitro Influence on Macrophage Activity and Cytokine-Like Protein Production in Fish

Histopathology of viremia-associated ana-aki-byo in combination with Aeromonas hydrophila in color carp Cyprinus carpio in Japan

A disease in which 'viremia-associated ana-aki-byo' is combined with an Aeromonas hydrophila infection currently occurs and is highly transmissible in color carp Cyprinus carpio in Japan. In the present study, to determine the interrelation between a corona-like virus and A. hydrophila, we conducted transmission trials by cohabiting naturally diseased carp with healthy carp with skin that had been slightly damaged artificially. Experimentally exposed fish successfully replicated the combination of a corona-like viral viremia and A. hydrophila infection. Diseased carp displayed scale-sac edema, ascites and exophthalmus adding to the formation of skin ulcers. In addition to pathological changes due to the corona-like virus infection, various changes due to the A. hydrophila infection occurred. Anasarcous skin lesions exhibited a separated epidermis, expanded scale-sacs, and an edematous dermis accompanied by hemorrhage and necrosis. The underlying lateral musculature was edematous and possessed markedly atrophied muscle fibers. Hepatocytes were either atrophied or swollen and had a granular appearance. Renal tubular cells showed vacuolar degeneration, cloudy swelling, necrosis and destruction. Hemosiderin deposition occurred within macrophages in the spleen and hematopoietic tissue, and within hepatocytes. Cardiac muscle fibers exhibited degeneration and necrosis accompanied by hemorrhage in the myocardium of heart. These changes appeared to be induced by bacterial toxins because bacterial cells did not directly invade these affected tissues.

Studies on the number and ingesting ability of thrombocytes in sick carps (Cyprinus carpio L.)

Carps suffering from acute form of brachiomycosis (age: 2 months), chronic form of brachionecrosis cyprinorum (age: 12 months), acute form of erythrodermatitis (age: 23 and 28 months) or chronic form of erythrodermatitis (age: 23 months) were studied. The level of thrombocytes in carps with acute forms of the diseases was significantly lower than that in healthy carps. The inverse pattern was observed in carps with chronic forms of the diseases. Carps suffering from the acute form of erythrodermatitis (age: 23 and 28 months) demonstrated no differences in the level of thrombocytes which could be related to age or body weight of the animals. Index of thrombocyte ingesting ability and percent of ingesting thrombocytes were lowered in sick carps as compared to carp of control groups. A significant decrease in the parameters was disclosed, however, only in carps with acute forms of the diseases. Similarly to the level of thrombocytes, neither did the level of their ingesting ability demonstrate any differences which could be related to the age of carps suffering from the acute form of erythrodermatitis

Resistance in carps (Cyprinus carpio L.) affected by a natural bacterial infection

Sick carps, affected by acute or chronic forms of erythrodermatitis (CE) were studied. The studies were aimed at obtaining pathophysiological data which would permit evaluation of clinical status and prognosis in carps of various age (23&amp;nbsp;or 28 months). This was approached by estimation of the number of carps and of dynamics of changes in nonspecific humoral and cell mediated resistance and in levels of selected serum proteins. The ingesting index of neutrophilic granulocytes and percent of neutrophilic granulocytes capable of ingesting proved significantly higher in sick carps as compared to the healthy carps, at every age of carps and form of the disease. Metabolic activity of neutrophilic granulocytes, expressed in the percentage of NBT+ cells was the same in carps with the acute CE form and in the control fish. On the other hand, in carps suffering from the chronic form of the disease a significant decrease in the number of cells was disclosed. NBT index and the amount of formazan in fishes with acute CE form demonstrated a decrease as compared to the corresponding values in control carps. Carps with the chronic CE form showed increases in the indices. The level of lysozyme (LZM) was higher in 23 or 28 months old carps with the acute CE form than in healthy fishes. In carps affected by the chronic CE form no significant differences in LZM levels were disclosed as compared to the healthy carps. MPO activity in the sick carps, particularly those with the acute form of CE, was significantly higher than in the control carps. On the other hand, serum globulin and total protein levels in the sick carps were lower than those in the control carps. The two indices showed a particularly extensive decrease in carps affected by acute form of the disease. When all the parameters estimated in fishes with acute CE forms were taken into account, no differences associated with age (23 or 28 months) were observed that could be related to tendencies and directions of the observed changes.

Flow cytometric analysis of proliferative responses of carp Cyprinus carpio peripheral blood leukocytes to mitogens and to the hemoflagellate Trypanoplasma borreli.

The activation of carp peripheral blood leukocytes (PBL) was analysed radiometrically and by means of flow cytometry (FCM) in order to compare the results obtained with both methods. The qualitative and quantitative FCM analyses of cellular morphology and viability resulted in a further characterisation of proliferative responses of carp PBL to Trypanoplasma borreli in vivo and in vitro. The lymphocyte population of PBL from T. borreli-infected carp exhibited a marked shift in forward scattered light (FSC; cell size). When PBL from healthy carp were stimulated with mitogens in vitro, a lymphoid population with increased FSC profiles was also observed. The number of these cells coincided to ratios of 3H-thymidine incorporation, recorded from corresponding cultures. Thus, it was concluded that the increase in size of stimulated lymphocytes could be due to blastogenic transformation. The advantage of the FCM procedure is that activation and proliferation of carp lymphocytes can be monitored without labelling the cells. Cocultures of mitogen-stimulated carp PBL and T. borreli revealed the ability of the parasite to suppress lymphocyte proliferation in vitro.

Yersinia ruckeri Septicaemia in Experimentally Infected Carp (Cyprinus Carpio L.) Fingerlings

The presence of Yersinia ruckeri, the causal agent of enteric redmouth disease (ERM) in salmonids and a few other freshwater fish, has so far been reported from a variety of sources including the intestine of healthy carp. Since there are no data on the pathogenicity of this bacterium for carp, 15 fingerlings were experimentally infected by intraperitoneal injection of about 5 x 10(5) cells. Thirteen injected fish were moribund or died within 4 days with septicaemic lesions. Two survivors were sampled on Day 28 after infection. Yersinia ruckeri was reisolated from the internal organs of all experimental fish. By histopathological examination moribund fish had generalised bacteriaemia with inflammation, degeneration and necrotic foci in kidney, liver and spleen, corresponding to findings described previously in ERM of rainbow trout. Survivors of challenge on Day 28 had a chronic disease characterised by prominent peritonitis and enteritis, exhaustion of the erythroid, granuloid and lymphoid components in haematopoietic kidney tissue as well as focal degeneration and necrosis in organs. These data indicate a high sensitivity of carp to intraperitoneal infection with a relatively low dose of Y. ruckeri.

Granulation staining and cytochemistry of peripheral blood leucocytes in healthy carp (Cyprinus carpio L.) I. Granulocytes

The cytochemistry and staining of granula in peripheral blood granulocytes in healthy carp (Cyprinus carpio L.) are described. Blood smears were stained for periodic acid-Schiff (PAS), peroxidase, oxidase, alkaline and acid phosphatase, α-naphthyl-acetate esterase, α-naphthyl-butyrate esterase, naphthol-AS-chloroacetate esterase (AS-D), naphthol-AS-acetate esterase and β-glucuronidase. Different granula types were shown by triazid-staining (eosinophil and neutrophil granula) and methylenblue-staining for basophil granulation. Toluidinblue-staining was used for basophil granulocytes. Lipids were shown by the Sudan-black-reaction. Four granulocyte subpopulations are described: neutrophil, heterophil, basophil and eosinophil granulocytes. Neutrophils possess all tested granula types, whereas heterophil and basophil granulocytes show only basophil granula. Neutrophils and heterophils show no activity of the tested esterases with the exception of AS-D. Only neutrophils were peroxidase-positive. Alkaline phosphatase and β-glucuronidase were not detected in granulocytes. Basophils and especially eosinophils were rarely found in peripheral blood.

Granulation staining and cytochemistry of peripheral blood leucocytes in healthy carp (Cyprinus carpio L.) II: Monocytes

Granulation staining and cytochemistry of peripheral blood monocytes in healthy carp (Cyprinus carpio L.) are described. Blood smears were stained for periodic acid-Schiff (PAS), peroxidase, oxidase, alkaline and acid phosphatase, α-naphthyl acetate esterase, α-naphthyl-butyrate esterase, naphthol-AS-chloroacetate esterase (AS-D), naphthol-AS-acetate esterase and β-glucuronidase. For representation of different granulations triazide-staining for eosinophil and neutrophil granules and aqueous methylere-blue staining for basophil granules were used. Lipids were shown by sudan-black-reaction. Monocytes showed only basophil granulation and weak lipid reaction. All tested enzymes were detected, with the exception of peroxidase. The PAS reaction for glycogen proof was negative.

Rapid detection of SVC virus antigen in infected cell cultures and clinically diseased carp by the enzyme-linked immunosorbent assay (ELISA)

Summary An ELISA was developed using a rabbit antiserum with high levels of binding antibody against SVC virus (SVCV). Modifications were made to the standard assay which increased the sensitivity and resulted in a rapid ELISA (rELISA) which could detect SVCV antigen in cell cultures and in extracts from infected carp in approximately 1 h. When other cell-grown fish rhabdoviruses were tested, pike fry rhabdovirus (PFRV) antigen cross-reacted in the rELISA but only at a low level. Virus antigen was detected by the rELISA in clinically and sub-clinically diseased carp during an SVC epizootic but the sensitivity of detection of subclinical levels of virus was not as great as that of isolation of infectious virus in cell culture. However, antigen was detected in some fish extracts which failed to yield infectious virus. Fresh extracts of organs from healthy carp were found to give false positive reactions in the rELISA. Kidney and liver extracts from these fish were found to contain a heat labile, non-specific binding factor and further modification of the rapid assay was undertaken which successfully reduced the effect of this factor.

Gill necrosis — a complex disease of carp

Gill necrosis is a widespread disease of common carp in Europe. Much effort has been concentrated in the investigation of its aetiology during the last 15 years. The results of a three-year research project are presented here and the role of bacteria is demonstrated. This paper shows the development of the disease and the relationship between environmental stress and bacterial invasion. Both healthy and diseased carp populations were monitored for the presence of Flexibacter columnaris. Gill necrosis was reproduced experimentally using Flexibacter columnaris isolates. Based on the bacteriological investigations, artificial infection and field monitoring, three stages of gill necrosis could be distinguished. The first stage is initiated and maintained by environmental stress (probably ammonia, pH, temperature or any toxins). At this stage, Flexibacter columnaris is seldom present on the gills if at all. The gills are pale or dark purple. The second stage starts when Flexibacter columnaris invades the damaged gill (only at water temperatures above 20°C). The infected part of the gill is necrotized and covered with a grey-white coating. The third stage occurs if the fish survives bacterial invasion. Flexibacter columnaris with its grey-white coating disappears and the site of the invasion remains distorted. Different stages of gill necrosis may appear in the same fish population.