Antiviral activities of Clinacanthus nutans (Burm.f.) Lindau extract against Cyprinid herpesvirus 3 in koi (Cyprinus carpio koi)

Cyprinid herpesvirus 3 (CyHV‐3) or koi herpesvirus (KHV) is a virulent viral infection in common carp and koi. The disease has caused global epizootic and economic loss in fish aquaculture and in the wild. Clinacanthus nutans (Burm. f.) Lindau is a well‐known medicinal plant used in Thai traditional medicine. Virucidal effects of the plant extract against human herpes simplex virus have been reported. In this study, C. nutans crude extract was tested for antiviral activities against CyHV‐3 in koi carp. Results showed effective antiviral activity against CyHV‐3 pre‐ and post‐infection. The 50% lethal concentration (LC₅₀) of extract was higher than 5 mg/ml. The 50% effective dose (ED₅₀) was 0.99 mg/ml, 0.78 mg/ml, 0.75 mg/ml and 0.71 mg/ml at 1, 2, 3 and 4 hr pre‐infection, respectively. The ED₅₀ from post‐infection tests was 2.05 mg/ml and 2.34 mg/ml at 0 and 24 hr, respectively. These results demonstrated that crude extract expressed antiviral activity against CyHV‐3 and can be applied as a therapeutic agent in common carp and koi aquaculture.     

Validation of a serum neutralization test for detection of antibodies specific to cyprinid herpesvirus 3 in infected common and koi carp (Cyprinus carpio)

Cyprinid herpesvirus 3 (CyHV‐3) is the aetiological agent of a serious infective, notifiable disease affecting common carp and varieties. In survivors, infection is generally characterized by a subclinical latency phase with restricted viral replication. The CyHV‐3 genome is difficult to detect in such carrier fish that represent a potential source of dissemination if viral reactivation occurs. In this study, the analytical and diagnostic performance of an alternative serum neutralization (SN) method based on the detection of CyHV‐3‐specific antibodies was assessed using 151 serum or plasma samples from healthy and naturally or experimentally CyHV‐3‐infected carp. French CyHV‐3 isolate 07/108b was neutralized efficiently by sera from carp infected with European, American and Taiwanese CyHV‐3 isolates, but no neutralization was observed using sera specific to other aquatic herpesviruses. Diagnostic sensitivity, diagnostic specificity and repeatability of 95.9%, 99.0% and 99.3%, respectively, were obtained, as well as a compliance rate of 89.9% in reproducibility testing. Neutralizing antibodies were steadily detected in infected carp subjected to restrictive or permissive temperature variations over more than 25 months post‐infection. The results suggest that this non‐lethal diagnostic test could be used in the future to improve the epidemiological surveillance and control of CyHV‐3 disease.     

Distribution of the introduced cyprinid herpesvirus 3 in a wild population of common carp, Cyprinus carpio L.

Cyprinid herpesvirus 3 (CyHV-3), which causes a lethal disease in common carp, Cyprinus carpio L., and koi, C. carpio koi , first occurred in Lake Biwa, Japan in 2004. To elucidate distribution of CyHV-3 in a wild common carp population, we conducted a PCR survey of CyHV-3 among such fish in Lake Biwa in 2006. Only 6% (1/18) of the common carp smaller than 300 mm were positive with PCR, whereas 31% (18/58) of fish larger than 300 mm were positive. To evaluate their past exposure to CyHV-3 infection based on the presence of antibodies, we also measured the levels of serum anti-CyHV-3 antibodies in the carp, using an enzyme-linked immunosorbent assay. None (0/26) of the fish smaller than 300 mm was positive for the antibodies, whereas 54% (33/61) of fish larger than 300 mm were positive. Of the antibody-positive individuals, 44% (14/32) were also positive by PCR strongly suggesting that wild common carp that survived infection become CyHV-3 carriers. Five individuals were positive by PCR but negative for antibodies indicating that their infection with CyHV-3 had occurred recently. These results suggest that transmission of CyHV-3 from carriers to naïve common carp is still occurring in Lake Biwa.     

Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV‐3) and possible latency of CyHV‐3 by temperature shift in the cells

A new cell line named CCF‐K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV‐3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV‐3‐infected CCF‐K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV‐3 was produced stably in CCF‐K104 cells over 30 viral passages. Our findings showed that CCF‐K104 is a useful cell line for isolation and productive replication of CyHV‐3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real‐time PCR showed that CyHV‐3 was present with low viral DNA loads, suggesting that CyHV‐3 may establish latent infection in CCF‐K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV‐3 genome arranged in a head‐to‐tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV‐3.     

Effects of graded levels of dietary myo‐inositol on non‐specific immune and specific immune parameters in juvenile Jian carp (Cyprinus carpio var. Jian)

The aim of this study was to evaluate the effects of myo‐inositol (MI) on non‐specific immune and specific immune defence in fish. A total of 1050 Jian carp (Cyprinus carpio var. Jian) (22.28±0.07 g) were randomly distributed into seven groups, of three replicates each, of feeding diets containing graded levels of MI (163.5, 232.7, 384.2, 535.8, 687.3, 838.8 and 990.3 mg MI kg^?1 diet). After a 60‐day growth trial, an infectious trial was conducted by injection of Aeromonas hydrophila for 17 days. Results showed that the red blood cell (RBC) and the white blood cell count were significantly increased with increasing MI levels up to 535.8 mg kg^?1 diet (P<0.05). The spleen index showed a tendency similar to RBC, whereas the head kidney index showed the inverse pattern (P<0.05). The phagocytic activity of leucocytes, haemagglutination titre, lysozyme activity, anti‐A. hydrophila antibody titre and immunoglobulin M, after being injected with A. hydrophila, were all improved with an increase in the MI levels up to 232.7–687.3 mg kg^?1 diet respectively (P<0.05). Myo‐inositol did not influence serum acid phosphatase activity and total iron‐binding capacity (P>0.05). These results suggested that MI could enhance non‐specific immune and specific immune responses in fish.     

d‐lysine can be effectively utilized for growth by common carp (Cyprinus carpio)

An 8‐week feeding trial was conducted to compare the effects of different lysine isomers on growth, nutrient utilization and metabolic enzyme activity in common carp (Cyprinus carpio). A basic experimental diet (the control group) with 30% of dietary protein from fish meal and the remaining 70% from soybean meal was used either with or without supplementation of d‐ lysine, l‐ lysine or dl‐ lysine, respectively. The total feed intake was significantly improved by supplementation with lysine regardless of isomeric form (P < 0.05). Supplementation with d‐ lysine or l‐ lysine (but not dl‐ lysine) generally increased the weight gain, specific growth rate, feed efficiency ratio and protein efficiency ratio. Dietary lysine supplementation generally increased the apparent digestibility of dry matter, crude protein and crude lipid, but no statistically significant differences were observed between the control group and the lysine‐supplement groups (P > 0.05). The d‐ amino acid oxidase and d‐ aspartate oxidase activities were generally higher in the liver, kidney and intestine of fish fed diet containing d‐ lysine compared to fish fed diet containing l‐ lysine. The lysine, protein and lipid gains in fish were markedly increased by the supplementation with d‐ lysine or l‐ lysine, and the lysine (r = 0.618, P < 0.05) and protein gains (r = 0.671, P < 0.05) were linearly related to dietary lysine intake. Lysine retention efficiency linearly decreased with the increase of dietary lysine intake (r = ?0.579, P < 0.05). These results indicate that common carp are capable of utilizing the supplemental crystalline d‐ lysine·HCl (4 g kg^?1 dry diet) for growth when the rest of dietary amino acids are protein‐bound forms.     

Menthol and 1,8‐cineole as new anaesthetics in common carp,Cyprinus carpio (Linnaeus, 1758)

This study describes anaesthetic efficacy of menthol and 1,8‐cineole in common carp, Cyprinus carpio, in comparison with eugenol. Common carp fingerlings were exposed to eugenol: 5, 10, 15, 25, 35, 50, 75, 100, 150 and 200 ppm; menthol: 5, 10, 15, 25, 50, 100, 200, 300, 400, 500 and 600 ppm; 1,8‐cineole: 100, 150, 200, 300, 400, 500, 600, 700 and 800 ppm. Induction time and recovery time were recorded. Results showed that menthol and 1,8‐cineole anesthetized the fish at higher concentrations compared to eugenol. The fish exposed to menthol showed common fish behavioural responses to anaesthesia, similar to eugenol. But, 1,8‐cineole‐exposed fish showed tail‐up swimming, which was not observed before. Also, 1,8‐cineole failed to completely cease muscle tone. Exposure to 200 ppm eugenol and 600 ppm menthol resulted in 40% and 20% mortality, respectively. Induction time was exponentially dependent on anesthetic concentrations. Recovery time was linearly correlated to eugenol and menthol, but not 1,8‐cineole concentrations. Recovery time was exponentially dependent on induction time in the fish anesthetized with eugenol and menthol, but not 1,8‐cineole. Menthol and 1,8‐cineole are recommended for carp anaesthesia. Menthol is capable to anesthetize common carp within 1–3 min at 118–512 ppm. Common carp anesthetized with 108–133 ppm menthol recovers within 5 min. 1,8‐cineole failed to anesthetize common carp within less than 150 s at 300–800 ppm concentrations. However, it anesthetizes carp within 3 min at 595 ppm concentration. Also, 1,8‐cineole is not recommended for fish surgery. Both menthol and 1,8‐cineole were less efficacious than eugenol.     

Examination of the early infection stages of koi herpesvirus (KHV) in experimentally infected carp,Cyprinus carpio L. using in situ hybridization

Koi herpesvirus (KHV) causes a highly infectious disease afflicting common carp and koi, Cyprinus carpio L. Various molecular and antibody‐based detection methods have been used to elucidate the rapid attachment and dissemination of the virus throughout carp tissues, facilitating ongoing development of effective diagnostic approaches. In situ hybridization (ISH) was used here to determine the target tissues of KHV during very early infection, after infecting carp with a highly virulent KHV isolate. Analysis of paraffin‐embedded tissues (i.e. gills, skin, spleen, kidney, gut, liver and brain) during the first 8 h and following 10 days post‐infection (hpi; dpi) revealed positive signals in skin mucus, gills and gut sections after only 1 hpi. Respiratory epithelial cells were positive as early as 2 hpi. Viral DNA was also detected within blood vessels of various tissues early in the infection. Notable increases in signal abundance were observed in the gills and kidney between 5 and 10 dpi, and viral DNA was detected in all tissues except brain. This study suggests that the gills and gut play an important role in the early pathogenesis of this Alloherpesvirus, in addition to skin, and demonstrates ISH as a useful diagnostic tool for confirmation of acutely infected carp.     

Detection methods of Cyprinid herpesvirus 2 infection in silver crucian carp (Carassius auratus gibelio) via a pORF72 monoclonal antibody

Cyprinid herpesvirus 2 (CyHV‐2) is the main pathogen responsible for causing haematopoietic necrosis disease in Carassius auratus gibelio. Although many nucleic acid‐based diagnostic methods have been applied, no stable and sensitive immunological diagnostic approaches have been reported. In this study, to detect CyHV‐2 in clinical samples using immunological methods, recombinant ORF72 protein (pORF72), encoded by the CyHV‐2 ORF72 gene, was used as a capture antigen to identify blood and tissues infected with CyHV‐2. First, ORF72 gene was amplified from the CyHV‐2 genome and cloned into a PGEX‐4t‐3 expression vector to produce pORF72 in Escherichia coli. The purified pORF72 was used as an immunogen to prepare monoclonal antibodies. The Western blotting assays revealed that the monoclonal antibody could specifically identify the pORF72. Furthermore, an immunohistochemical protocol and a blood smear method were established to detect CyHV‐2 in carps. The results indicate that the monoclonal antibody against pORF72 could be utilized as an effective detection tool for haematopoietic necrosis disease in Carassius auratus gibelio.     

The effect of varying dietary digestible protein and digestible non‐protein energy sources on growth,nutrient utilization efficiencies and body composition of carp (Cyprinus carpio) evaluated with a two‐factorial central composite study design

To determine optimized dietary nutrient supply for common carp (Cyprinus carpio), nine isoenergetic diets were formulated according to a two‐factorial central composite design on digestible basis. Digestible protein contents (DP) in diets ranged from 208.8 to 251.2 g/kg, and digestible fat‐to‐digestible carbohydrate energy ratios (DEF/DEC) varied from 0.79 to 1.21. Carp were fed four times a day at a daily feeding rate of 2.5 g/kg body weight for 56 days. Optima of responses were determined by applying the standard least squares method according to response surface methodology. All diets resulted in excellent performance with feed conversion ratios lower than 1 and digestible protein efficiency ratios (PER_dig) higher than 4.6. For most parameters, calculated extreme values were minimum points, except for FCR, retention efficiencies of digestible arginine (RetArg_dig) and of digestible methionine (RetMet_dig), being maximum points, and retention efficiencies of digestible isoleucine, digestible sulphuric amino acids and digestible lysine, being saddle points. Highest growth and feed utilization efficiency were graphically determined for a diet containing 208.8 g/kg DP/1.21 DEF/DEC. Body lipid was optically increased for 251.2 g/kg DP/0.79 DEF/DEC in comparison with 208.8 g/kg DP/1.21 DEF/DEC.     

Potential applications of biological filtration media in changing water properties and subsequent effects on common carp (Cyprinus carpio) in recirculating aquaculture systems

This work aims to develop a suitable aquaculture system to solve issues pertaining to water quality in recirculating aquaculture systems (RAS) thereby improving fish growth performance and their feed conversion ratio (FCR). It is known that the efficiency of nutrient decomposition depends on the amount and species of bacteria present in the filter, and these can in turn be regulated by the pH. Our study shows that the significant increase in pH from 7.45 to 7.51 and 7.47 is achieved after the water was filtered through sawdust and wood wool respectively. At the same time, nitrite concentrations were found to have significantly decreased from 0.06 ppm in the control to 0.01 ppm in the sawdust treatment. Changes in the physical–chemical properties of treated water affect the biological properties of the organisms. It was found that the average final body weight for fish in the wood wool treatment 30.96 g and fish in the sawdust treatment 31.44 g was significantly higher compared to that for the fish in the control treatment 28.42 g. The use of sponge, wood wool or sawdust resulted in significantly lower FCR and therefore better fish productivity in RAS. At the same time, the red blood cell count, plasma urea, plasma creatinine, plasma albumin, and GOT and GPT were significantly higher in fish in the plastic, sponge, wood wool or sawdust treatment compared to the control treatment. In summary, this study demonstrates the benefits of using sponge, wood, wool and sawdust as biofilter media for aquaculture.     

A novel testicular degenerative condition in a wild population of the common carp Cyprinus carpio (L)

Gonad abnormalities can restrict or completely block reproductive capability of individuals and in some case that of their populations. Here, we describe a novel testicular degenerative condition of non-germ cell origin with a high prevalence (up to 22.1% of the population) in a wild population of carp. Based on gross morphology, and microscopic and cellular examinations, the condition shows progressive severity which could be categorized into low, mild, severe and complete. In early stages of the condition, an abnormally increased proliferation (11-fold) of the Sertoli cell occurred, followed by degenerative cell death of all testicular cells, resulting in fluid-filled vesicles in the later stages. This initial uncontrolled proliferation of Sertoli cells suggests that the condition could be triggered by malignant pathways; however, the observed subsequent apoptosis of all testicular cells en masse, rendering the animals “sterile,” appears unique. Observations, to date, indicate that this condition is specific to male carp and not present in other species of fish sharing the habitat. High prevalence of the condition allowed comparative evaluation between affected individuals, an aspect likely to facilitate future studies, including elucidation of the cause, robust testing of therapies and practical applications such as management of feral carp populations.     

The study of antioxidant enzymes and immune‐related genes expression in common carp (Cyprinus carpio) fingerlings fed different prebiotics

The aim of the present study was to study the effects of different prebiotics (galacto‐, fructooligosaccharide and inulin) on immune response and oxidative stress of common carp (Cyprinus carpio) fingerlings at the molecular level. A total number of 240 fish (13.85 ± 0.85 g) were supplied and randomly stocked in twelve fiberglass tanks (20 specimens per tank). Fish were fed a basal formulated diet (Control) or basal diet supplemented with equal level (2%) of different prebiotics (four treatments repeated in triplicated) for 8 weeks. At the end of feeding trial, the expression of immune‐related genes (interleukin 1 beta [IL‐1β], IL‐8, IL‐10, lysozyme [LYZ], tumour necrosis factor alpha [TNF‐α] and transforming growth factor beta [TGF‐β]) were determined in head kidney and intestine tissues and the expression of antioxidant‐related genes (glutathione S‐transferase [GST‐α], glutathione reductase [GR] and glutathione peroxidase genes [GPX]) were studied in intestine. The results revealed that dietary administration of prebiotics modulated the expression of immune‐related genes and the degree of expression was affected by the type of prebiotics and the organ that was used for analyses. Also, evaluation of antioxidant genes expression showed that GSTα and GR expression levels increased as a result of feeding common carp with the prebiotics. According to these findings, it can be concluded that feeding on different prebiotics had altered effects on the expression of immune and antioxidant‐related genes.     

Effects of α‐ketoglutarate on the growth performance,amino acid metabolism and related gene expression of mirror carp (Cyprinus carpio)

A 70‐day feeding experiment was conducted to assess the effects of α‐ketoglutarate (AKG) supplementation in different protein sources of diets on the growth performance, nitrogen metabolism and related gene expression of mirror carp. Two kinds of protein diets (soybean meal protein source and fish meal and soybean meal compound protein source) and two levels of AKG (0 g kg^?1 and 15 g kg^?1, respectively) were utilized. Each experimental diet was fed three times a day with five replicates of 20 fish per tank with an average individual weight of 217.93 ± 0.78 g. The results indicated that AKG significantly improved the protein efficiency ratio and decreased the feed conversion rate (FCR) (p < .05). Soybean protein significantly decreased the weight gain rate and increased the FCR (p < .05). In addition, diets with AKG significantly improved the concentration of glutamine and glutamic acid (Glu) in the foregut and hindgut (p < .05), as well as the glutamine synthetase (GS) activity in the foregut (p < .05). Soybean protein significantly decreased the concentration of Glu and GS activity in the hindgut (p < .05). AKG significantly improved the contents of threonine, alanine, proline and essential amino acid in muscle (p < .05). Dietary supplementation with AKG significantly improved GS gene expression in the intestine and reduced Rhesus glycoprotein (Rhag and Rhbg) gene expression in the gill (p < .05). From the results, we know that a diet with AKG is beneficial for the growth and amino acid metabolism of carp.