Mass mortality associated with koi herpesvirus in wild common carp in Canada

Koi herpesvirus (KHV) was identified as being associated with more than one mortality event affecting common carp in Canada. The first was an extensive mortality event that occurred in 2007 in the Kawartha Lakes region, Ontario, affecting Lakes Scugog and Pigeon. Fish had branchial necrosis and hepatic vasculitis with an equivocal interstitial nephritis. Several fish also had branchial columnaris. Subsequent mortality events occurred in 2008 in additional bodies of water in south-central Ontario, such as Lake Katchewanooka and outside of Ontario in Lake Manitoba, Manitoba. Koi herpesvirus was detected in fish submitted for examination from all of these lakes by polymerase chain reaction (PCR), and sequence of the PCR product revealed 100% homology to KHV strains U and I. Real-time PCR analysis of KHV-infected wild carp revealed viral loads ranging from 6.02×10(1) to 2.4×10(6) copies μg(-1) host DNA. This is the first report of KHV in Canada.     

Seminoma in a koi carp Cyprinus carpio: histopathological and immunohistochemical findings

A spontaneous seminoma in a 3 yr old male koi carp Cyprinus carpio L. is described. The animal, presenting a symmetric abdominal enlargement, showed a celomatic multinodular, white-yellowish and firm mass that infiltrated the liver and the intestine wall. Histologically, the neoplasm was non-encapsulated and poorly demarcated, showed invasive growth and was characterized by a lobular architecture, subdivided by abundant fibro-connective septa. Large necrotic and calcified areas together with small aggregates of residual spermatids were present. We diagnosed a classical seminoma with a diffuse pattern. Neoplastic cells cross-reacted with vimentin, placental alkaline phosphatase, and c-KIT. An immunohistochemical phenotypization of the tumor was performed to exclude other celomatic neoplasms and to compare this seminoma with those reported in mammals and humans.     

Initial characteristics of koi herpesvirus and development of a polymerase chain reaction assay to detect the virus in koi,Cyprinus carpio koi

Since 1998, episodes of mass mortality have occurred in populations of common carp Cyprinus carpio carpio in Israel and in populations of koi Cyprinus carpio koi in Israel and the USA. A herpesvirus isolated from infected fish has been shown in experimental studies to induce disease and mortality similar to those observed in outbreaks at infected farms. Initial characteristics of the virus show that it is clearly different from Herpesvirus cyprini (CHV), the most commonly known herpesvirus from cyprinid fish. The koi herpesvirus (KHV) has 31 virion polypeptides. Twelve of the virion polypeptides of KHV have similar molecular weights to those of CHV and 10 are similar to those of channel catfish virus (CCV). Both virion polypeptide and restriction fragment length polymorphism analyses of genomic DNA showed that the first KHV isolates from Israel and the USA were identical. In contrast, the genomic DNA restriction fragments clearly distinguish KHV from CHV and CCV. A polymerase chain reaction (PCR) assay to detect the virus in koi tissues was developed with sequences obtained from 1 restriction fragment of KHV DNA. The PCR assay effectively detected a 484 base pair sequence from KHV but did not amplify genomic DNA from either CHV or CCV. The PCR assay detected as little as 1 pg of KHV DNA mixed with 100 ng of host DNA. Viral sequences were amplified from koi obtained from field collections and from koi that were experimentally exposed to 10(2) TCID50 ml(-1) of KHV via the waterborne route. All KHV exposed fish dying of infection between 8 and 10 d post exposure or surviving to 14 d post exposure were found to be positive by PCR, while unexposed control koi were all negative. The assay also showed the presence of KHV DNA in tissues of koi obtained from farms in Israel. The PCR assay should assist virus isolation procedures and histologic and electron microscopic analyses now commonly used to detect KHV infection. Current studies are examining the possibility of using the PCR to detect KHV DNA in live fish and the relative sensitivity and specificity of the KHV PCR assay compared with other diagnostic tests.     

Efficacy of quinine against ichthyophthiriasis in common carp Cyprinus carpio

Ichthyophthiriasis, caused by Ichthyophthirius multifiliis, is an economically important worldwide parasitic disease that infects all freshwater fish. Since the banning of malachite green for use in food fish, there has been a great need for alternative therapeutants. The goal of this study was to investigate the efficacy of quinine against I. multifiliis. Parasite developmental stages from our laboratory-established life cycle in rainbow trout Oncorhynchus mykiss were exposed to quinine in vitro, and a dual fluorescent staining technique was used to allow a clear distinction between viable and damaged parasites. Furthermore, the effect of quinine was assessed in vivo by oral administration and intraperitoneal injections in common carp Cyprinus carpio. The results of the in vitro experiments proved quinine to be effective against the parasite. Quinine injected at a dosage of 60 mg kg(-1) body weight resulted in a significantly lower number of trophonts. In contrast, in-feed trials did not show a significant reduction of trophonts after treatment commencing 1 d after infection with concentrations of up to 20 g quinine kg(-1) feed for 3 d. After a 14-d treatment at concentrations of up to 10 g quinine kg(-1) feed prior to theront exposure, there was also no significant difference in parasite numbers between treated and control groups. The results of oral versus parenteral application of quinine indicate that the substance is not completely absorbed from the intestinal tract of common carp. However, medicated feed containing higher concentrations of quinine was less readily accepted by the fish, presumably due to the bitter taste.     

Spontaneous diploidization of maternal chromosome set in ornamental (koi) carp, Cyprinus carpio L.

An unusually high frequency of spontaneous diploidization in a maternal chromosome set (SDM) was discovered in one ornamental carp (koi) female in an experiment on these fish for induced gynogenesis. Spontaneous appearance of diploid embryos in the gynogenetic offspring (intact eggs × irradiated sperm) and appearance of triploids among control fish (intact eggs × intact sperm) obtained from koi female and males of edible carp indicated spontaneous diploidization of maternal chromosomes (SDM). Possible cytological processes causing this phenomenon are discussed.     

Effects of a parasite-induced nephritis on osmoregulation in the common carp Cyprinus carpio

Carp Cyprinus carpio infected with the haemoflagellate Trypanoplasma borreli undergo progressive nephritis associated with a destruction of approx. 40% of the nephric tubules. In an attempt to analyse the effect of the nephritis on the osmoregulation of affected carp, the clinical chemical properties of plasma and urine samples were analysed. Parasitised carp excreted greater amounts of electrolytes in their urine than uninfected carp which excreted highly diluted urine with an osmolality of about 10% of plasma osmolality. During the course of the infection, urine osmolality increased up to 26% of plasma osmolality by Day 21 post-infection (p.i.). The plasma:urine ratio of Na+ also increased, while concomitant losses of Mg2+, Ca2+, K+ and inorganic phosphate were less pronounced. Infected carp were able to maintain a normal solute balance in their plasma. Plasma hydration (indicated by decreased protein contents) occurred on Day 21 p.i. Our data indicate that in T. borreli-infected carp, reabsorption processes of the distal renal tubule were disturbed, while secretory and absorption processes in the proximal tubule appeared to be less affected. In addition, infected carp were able to compensate their increased ion losses, probably by (energy-consuming) active absorption processes. The energy budget of infected carp was additionally affected by a substantial direct consumption of plasma glucose by the parasite.     

Determination of the surface area of common carp,Cyprinus carpio L.

A wrap method adaptation combined with AutoCAD2005 and Scion Image for Windows were used to determine the surface area of a fish. Compared with the corresponding r² and F of many models, the most accurate formula: S = 752.15W^0.675 (r² = 0.999, F = 18362.94, P < 0.0001) for estimating the surface area of common carp was obtained. Similarly, the fin formula: S = 1834.12W^0.708 (r² = 0.992, F = 2690.47, P < 0.0001) was also obtained for the same purpose. It was proven that these two formulae gave good estimates of surface and fin areas of four strains of common carp: Yellow‐river carp, fancy carp, mirror carp and Xingguo red carp.     

Cadmium-binding to transferrin in the plasma of the common carp Cyprinus carpio

In this study, it was investigated by autoradiography with radioactive cadmium after Western blotting of two-dimensional electrophoresis gels, to which proteins cadmium is mainly bound in plasma of common carp Cyprinus carpio. The obtained results demonstrate that in carp plasma, cadmium is primarily bound to two high molecular weight proteins. Relative small amounts are bound to a protein with M(r) approximately 60000. The other metal-binding protein, with M(r) approximately 70000 and pI approximately 6.7 was identified as transferrin. The conditional equilibrium constants for the binding of cadmium ions to the two metal-binding sites of this protein were calculated as logK(1)=5.40+/-0.12 and logK(2)=4.66+/-0.21, which are comparable to those of human transferrin under the same experimental conditions. Transport of cadmium in plasma of carp was found to be different from that of brown trout Salmo trutta and man, where cadmium is mainly bound to albumin and transferrin. The prominent binding of cadmium to transferrin can be explained by the absence or at least the very low concentrations in which albumin is present in carp plasma.     

Ultrastructural identification of a herpes-like virus infection in common carp Cyprinus carpio in Korea

We report on a herpes-like virus, which was found to be associated with mass mortality of common carp Cyprinus carpio for the first time in Korea in 1998. The external signs of infection in moribund fish were darkened coloration and severe branchial necrosis in the gill. Transmission electron microscopy revealed the presence of herpes-like viruses in spleen tissue. Infected spleen cells showed hypertrophied nuclei and degeneration. Numerous nucleocapsids of about 82 nm in diameter were found within the nucleus and cytoplasm of infected cells, and extracellular enveloped particles were also observed. We conclude that this virus was a likely significant cause of the high mortality of common carp in Korea in 1998.     

Gonadotropin response to GnRH during sexual ontogeny in the common carp, Cyprinus carpio

This study was designed to reveal whether gonadotropic response to GnRH in the common carp (Cyprinus carpio) changes during sexual ontogeny and whether the response of FSHbeta and LHbeta subunits is uniform or differential. The study comprised fish at the following stages: juveniles (4-month-old females with primary oocytes and early spermatogenic males); maturing (9-month-old previtellogenic females and advanced spermatogenic males); and mature (16-month-old postvitellogenic females and spermiating males). Fish were injected with superactive salmon GnRH analogue (sGnRHa; 25 microg/kg) and blood was sampled 6, 12 and 24 h later for cGtH (LH) and sex steroid levels. Pituitaries were taken for determination of FSHbeta and LHbeta mRNA levels by slot-blot hybridization and for cGTH content in the same glands by radioimmunoassay (RIA). Values were compared with the levels prior to sGnRHa administration and with control fish sampled at the same intervals. Juvenile fish did not respond at all to sGnRHa. In maturing females, FSHbeta mRNA increased by >300%, while that of LHbeta increased by 200%. In maturing males, FSHbeta mRNA did not change and only a slight increase occurred in that of LHbeta. In 16-month-old postvitellogenic females, there was no response of FSHbeta mRNA, while that of LHbeta dramatically increased. In spermiating males of the same age, mRNA of both FSHbeta and LHbeta increased following sGnRHa injection. Immunoreactive cGtH was present in the pituitary and plasma of all fish examined, but in juveniles it did not change following sGnRHa injection. In maturing and mature fish of both genders, sGnRHa administration was followed by a marked increase in circulating cGtH, concomitant with a decrease in its pituitary content, indicating the limited amount of the hormone stored in the gland. In conclusion, the response of the gonadotropin subunit mRNAs in the common carp was found to be differential and dependent on the gender and the phase of sexual ontogeny.     

Long-term cortisol treatment inhibits pubertal development in male common carp, Cyprinus carpio L

The onset and regulation of puberty is determined by functional development of the brain-pituitary-gonad (BPG) axis. Stress has been shown to interfere with reproduction and the functioning of the BPG axis. The response to chronic and severe stress may require much energy and force the organism to make adaptive choices. Energy that is normally available for processes like growth, immune response, or reproduction will be channeled into restoration of the disturbed homeostasis. Cortisol plays a key role in the homeostatic adaptation during or after stress. In the present study, immature common carp were fed with cortisol-containing food pellets covering the pubertal period. We showed that cortisol caused an inhibition of pubertal development, by affecting directly or indirectly all components of the BPG axis. The salmon GnRH content of the brain was decreased. Luteinizing hormone- and FSH-encoding mRNA levels in the pituitary and LH plasma levels were diminished by long-term cortisol treatment, as was the testicular androgen secretion. Testicular development, reflected by gonadosomatic index and the first wave of spermatogenesis, was retarded.     

The effect of digested sludge on the common carp, Cyprinus carpio L.

The effects of sewage sludge on the survival of the common carp, Cyprinus carpio, were investigated. It was noted that, when the concentrations of the sludge used for rearing carp exceeded 0.6%, the fish died within 42 days, whereas those reared in 0.2% sludge remained alive and healthy during the trial period. The body and the gills of the dead fish were covered by a veil-like film which looked like coagulated mucus and was formed by the heavy metal ions from the sludge reacting with some constituents of the mucus secreted by the gill. The histopathological symptoms of the various organs further demonstrated that internal injury in liver and kidney was also an important feature of the intoxicants as well as external damage, especially the particles observed around the gills. It was concluded that the optimum concentration of sludge to any species of fish should be determined by testing a series of concentrations before using it as a supplementary fish feed or fish pond fertilizer.     

Interactions of common carp (Cyprinus carpio) with benthic crayfish decapods in shallow ponds

Introduction of common carp (Cyprinus carpio) for aquaculture increased in recent decades. This fish is now established in many new water systems creating interactions with native species. Some of these interactions have been partly understood, but most of them remain unknown. For instance, in shallow ponds of central Mexico, populations of crayfish (Cambarellus montezumae) are reduced with high carp densities, but little is known about the mechanisms that lead to this depletion. Gut analysis showed that carp ate mostly detritus, small invertebrates, plant tissues and seeds, reducing the possibility of predation as a main cause of crayfish population reduction. Field and experimental data suggest that the effect of carp on crayfish is associated with habitat depletion. Submerged macrophyte Potamogeton pectinatus and the algae Cladophora glomerata are important components in crayfish habitat, and their coverage in the water system is affected by carp presence. A second effect of carp on crayfish populations is associated with the alteration of crayfish behaviour. Crayfish displacement speed increased significantly in the presence of carp.     

The eye lens weight and age in the common carp, Cyprinus carpio L.

The validity of the use of the weight of dried eye lenses as an indicator of age in the common carp, Cyprinus carpio L., is tested. There is a significant difference between males and females for fish of two and three years old. Otherwise no significant difference is noted between sexes. The linear relationship between the dry weight of eye lenses and age + 1 are: In Y + 1= 0.5202 + 16.4928 X ( r = 0.93) for female and ln Y + 1=0.4276 + 16.7364 X ( r = 0.94) for male.
The method for determining age is valuable for fish until three years old. However for older fish, in spite of a correlation coefficient between lens weight and age and even with a significant difference between annual groups, the age determination may be misleading due to the overlap in lens weights. In conclusion therefore this method may not be useful for back calculation of growth, for production estimates or studies in which accurate estimates of age are needed.     

Microridges of oral mucosal epithelium in carp,Cyprinus carpio

Summary The surface of carp oral mucosa is characterized by various patterns of microridges about 0.3 m wide, 0.1 m high, and of various lengths. To elucidate the derivation and function of these microridges, the oral epithelium was examined by light- and electron microscopy. Microridges were present only on the surfaces of the superficial cells. Therefore, microridges on renewed superficial cells are presumed to be formed after old superficial cells have been discarded, and the various patterns of microridges found on the cell surface appear to indicate the progress of their development. In thin sections, the outer leaflet of the plasma membranes of microridges stained strongly with ruthenium red, and the underlying cytoplasm was packed with many fine filaments. The superficial cells contained many secretory vesicles that were PAS-positive but Alcian blue-negative at pH 2.5 and pH 1.0. However, after sulfation the vesicles gave a positive reaction with toluidine blue. These vesicles are secreted by exocytosis at the free surface of the cells. After release, the membranes of the vesicles are thought to be utilized for formation of microridges. On the basis of these observations, the possible function of microridges is discussed.This study was supported by grants from the Ministry of Education, Science and Culture, Japan     

Analysis of genes expressed in head kidney of common carp Cyprinus carpio L treated with cortisol

We analyzed genes expressed from head kidney of common carp Cyprinus carpio L. treated with cortisol. The results of single-pass sequencing of expressed sequence tags (ESTs) from 188 clones (AU240288–AU240367, AU301120–AU301227) from kidney cDNA are presented. One-hundred-twenty-seven clones (67.6%) were completely unknown and are likely to represent newly described genes, whereas 61 clones (32.4%) were identified based on matches to sequences in the database. The putative genes contain several ribosomal proteins, cytochrome oxidase subunits. Immune related cDNA clones identified from kidney were immunoglobulin light chain (n=4), FK506/rapamycin-binding protein (FKBP), CXC chemokine receptor type 4, complement factor B/C2-A3, peptidylprolyl isomerase (cyclophilin; Cyp)-like1, cyclophilin S1, heat shock-70 kDa protein-4, stress-activated protein kinase-3 (n=2). FKBP and cyclophilin genes expressed in normal tissues (head kidney, spleen, liver, brain and heart). Expression of FKBP and cyclophilin genes were not detected in liver, brain and heart when treated with cortisol for 16 h.     

Involvement of fibronectin during epiboly and gastrulation in embryos of the common carp,Cyprinus carpio

Summary The present report firstly describes a pilot study in which, during early development of embryos of the common carp, Cyprinus carpio, the cellular adhesion to fibronectin (FN) was blocked by administration of GRGDS peptide (which binds to the FN-receptor). As this treatment resulted in developmental aberrations, suggesting a functional role for FN, the major part of the work was focussed on the distribution of reactivity of anti-FN antibodies during epiboly and gastrulation. GRGDS treatment had a concentration dependent effect on development. Incubation of embryos in 1.5 mg/ml from the 32-cell stage onwards caused a retardation of epiboly, which did not proceed beyond 60%. The embryos did not show involution, as was confirmed by histological study. These preliminary results suggest that FN is involved in both epiboly and gastrulation of carp embryos. During cleavage, no specific extracellular binding of anti-FN antiserum could be observed. However, binding to a number of cell membranes took place from early epiboly onwards. After the onset of gastrulation, we observed a gradually increasing number of the deepest epiblast cells, showing immunostaining on part of their surface, facing the yolk syncytial layer (YSL) or the involuted cells. During early epiboly, anti-FN binding was restricted to areas in front of the migratory hypoblast cells. Later on, binding was found at the border of hypoblast and epiblast cells. At 100% epiboly, some contact areas of epiblast and hypoblast showed a discontinuous lining of reactivity, whilst other areas appeared devoid of anti-FN binding sites. The results indicate that FN is involved in the migration and guidance of hypoblast cells during gastrulation in carp. Correspondence to: P. Gevers     

Occurrence of Atractolytocestus huronensis (Cestoda, Caryophyllaeidae) in German pond-farmed common carp Cyprinus carpio

Up to now, the caryophyllid cestode Atractolytocestus huronensis Anthony, 1958, a parasite of common carp, has attracted little attention in Germany. Based on recent publications from the Czech Republic and Hungary, it appears probable that this cestode may be increasingly common in Germany. There is a strong connection between the occurrence of A. huronensis and imports of common carp from the Czech Republic and southern Germany. Although in most cases no clinical alterations in parasitized carp have been observed, care should be taken to avoid further dissemination and to prevent possible losses in commercial pond farming.     

The biology of the common carp, Cyprinus carpio L. in the Camargue, southern France

Carp were sampled monthly during one and a half years in the Etang du Vaccarès, Camargue, southern France using fyke nets. Densities of carp in temporary marshes and canals were determined using rotenone. Carp is the dominant species in biomass in the fresh and brackish waters (14%0) with densities from 11–58 kg/ha in temporary marshes and up to ten times greater in the canals. Scales were used to determine age and growth of 201 carp. The relative condition factor of female and male mature and unsexed fish are not statistically different throughout the year and follow the variations of gonadal development in spring. The best predictive equation of fecundity, Log F = 7.03504 + 0.83829 log ovary weight, was found using a stepwise multiple regression. The sex-ratio in the temporary marshes (♂ : ♀; 1.66–1.76) is greater than the general sex-ratio of the monthly samples (0.67). The diet of carp is characterized by the presence of large numbers of seeds and the absence of large benthic invertebrates. Predation by fish-eating birds and carnivorous fish is minimal, and the annual drying of many marshes is considered the main mortality factor., Accepted 22 July 1980