Susceptibility of koi × crucian carp and koi × goldfish hybrids to koi herpesvirus (KHV) and the development of KHV disease (KHVD)

Hybrids of koi, Cyprinus carpio × crucian carp, Carassius carassius and koi × goldfish, Carassius auratus, proved to be susceptible to koi herpesvirus (KHV, syn. CyHV‐3) and developed KHV disease (KHVD). While hybrids of koi × goldfish were partly resistant to mortality following infection by immersion, most koi × crucian carp hybrids died after bath infection. KHV DNA was detected in dead fish but also in all surviving animals by different polymerase chain reactions (PCRs). According to these results, hybrid crossbreeding does not seem to prevent severe losses associated with KHV in terms of inducing KHVD. The present study showed severe losses after a waterborne KHV infection of between 35% and 100% in koi × goldfish and koi × crucian carp hybrids as well as in SPF carp.     

Correlation of hematology and plasma chemistry levels in silver crucian carp Carassius langsdorfii

ABSTRACT: The correlation between hematology and plasma chemistry was examined in natural silver crucian carp Carassius langsdorfii. Three microsatellite DNA markers identified five clonal lines. Blood was obtained from the caudal vessels and analyzed using automatic analysis systems. The correlation between several parameters including erythrocyte count (RBC), mean corpuscular hemoglobin concentration (MCHC), alkaline phosphatase (ALP), plasma glucose (Glu) and total protein (TP) was assessed by liner regression. Hemoglobin (Hgb) and hematocrit (Hct) as a function of the erythrocyte count, hematocrit as a function of the hemoglobin, mean corpuscular hemoglobin (MCH) as a function of the mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC) as a function of the mean corpuscular hemoglobin, and total protein as a function of triglyceride (TG) were positively correlated for the five clonal lines. Different regression slopes ( b -value) of RBC to Hgb and Hct were found among clonal lines, while no such differences were found among slopes of the remaining regression equation. This phenomenon suggests that the significantly different slopes among parameters, which indicated low genetic influence at the each parameter, clearly detected the genetic influences as the compound effect of these parameters.     

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.     

银鲫与普通鲫群体遗传结构的比较分析

利用15个微卫星多态性分子标记,对黑龙江省方正县双龙水库和双凤水库两个水体的雌核发育银鲫(Car-assius auratus gibelio)和二倍体普通鲫(Carassius auratus(L))的群体遗传多样性进行比较分析。结果显示:平均每个多态性位点获得银鲫10.39个有效等位基因,普通鲫11.82个有效等位基因;双龙水库三倍体银鲫和二倍体普通鲫的平均观测杂合度分别为0.6035和0.6300,双凤水库中的三倍体银鲫和二倍体普通鲫的平均观测杂合度为0.5311和0.5885;相似性与遗传距离分析结果显示:相似性最大的是双凤水体内的银鲫和普通鲫(0.8127),远大于两个水体间的银鲫群体间的相似性。结果表明,银鲫与普通鲫存在着明显的遗传物质交流,较高的遗传相似性不仅与传统的银鲫起源于普通鲫的观点一致,也暗示银鲫与普通鲫存在经常性地遗传物质交流。     

Availability of culture filtrate protein-10 (CFP-10) secreted from Mycobacterium pseudoshottsii for mycobacteriosis diagnosis in ginbuna crucian carp Carrasius auratus langsdorfii

Mycobacteriosis in cultured fish is a challenge for the aquaculture industry worldwide. Treatment by chemical administration is difficult and no effective vaccine has been developed. Therefore, detection and isolation by early diagnosis are important for prevention of the spread of the disease. In mammals, interferon gamma release assays have been established for detection of tuberculosis; these tests are based on the delayed-type hypersensitivity (DTH) response against culture filtrate protein-10 (CFP-10) and the 6-kDa early secreted antigen target (ESAT-6) of Mycobacterium tuberculosis. On the other hand, little is known about the fish immune response against the ESAT-6 and CFP-10 proteins of mycobacteria, although these responses should find application in the diagnosis of mycobacteriosis in fish. In the present study, we identified ESAT-6 and CFP-10 from Mycobacterium pseudoshottsii and cloned the corresponding genes. Intraperitoneal injection of the corresponding DNA plasmid constructs in ginbuna crucian carp yielded increased expression of the fish interferon-γ1-1-encoding gene (IFN-γ1-1). In contrast, IFN-γ1-1 expression accompanied by DTH response was observed only in the CFP-10-DNA plasmid-injected fish. Furthermore, fish that had been prophylactically injected with CFP-10-DNA plasmid exhibited increased survival of M. pseudoshottsii infection. Taken together, these results suggested that CFP-10 may facilitate diagnosis of mycobacteriosis.     

New detection of Cyprinid herpesvirus 2 associated with mass mortality in colour crucian carp (Carassius auratus), in China

During October 2016, a mass mortality of colour crucian carp (Carassius auratus), which the affected fish were lethargic, inappetence and anoxic, was occurred in a fish farm located in Chengdu, Sichuan province, China. To elucidate the aetiology of this outbreak, histological and electron microscope examination, molecular investigation were conducted. Pathologic examination revealed multi foci necrosis on haematopoietic organs, gills, hearts and pancreas. Transmission electron microscopy observations exhibited sphere herpesvirus‐like particles distributed amongst the tissues of gill, spleen and kidney. Molecular analysis is verified that the causative agent of this outbreak was Cyprinid herpesvirus 2 (CyHV‐2). This report first report CyHV‐2 in colour crucian carp, which increases the concern about damage of CyHV‐2 and its potential role in species.     

Thermal resistance traits of the clonal silver crucian carp Carassius langsdorfii and evaluation of these traits using primary culture cells

The thermal resistance traits of three clonal lines of the silver crucian carp Carassius langsdorfii (SCC-1, -2 and -3) were investigated. Individual juvenile fish reared at 20°C were exposed to thermal stress at 36.5 ± 0.5°C and their death times during this exposure were measured. The death times of SCC-2 and SCC-3 fish were 66.6 ± 31.2 and 144.7 ± 49.8 min, respectively. In contrast, all SCC-1 fish survived under these conditions. Furthermore, the thermal sensitivity of primary culture cells from each clone was determined at 37, 40 or 43°C by the Trypan blue assay. Under all treatment conditions, the thermal sensitivity of the SCC-1 primary cultures was lower than those of the others. These results estimated the correlation between the in vivo and in vitro thermal resistances. Therefore, the use of primary culture cells to evaluate thermal resistance could be a useful method of selection breeding.     

雄核发育异育银鲫及其初步应用

用鲫（２ｎ＝１００）卵与异育银鲫（３ｎ＝１５６）正常精子进行人工授精,产生了部分的雄核发育异育银鲫（３ｎ＝１５６）。池塘中饲养的雄核发育能银鲫性腺不仅能成熟,而且也能生殖。它与父本异育银鲫回交可以产生回交子一代（Ｂ１）。雄核发育异育银鲫、回交子一代异育银鲫与父本异育银鲫非常相似。它们均具有相同的染色体数目（３ｎ＝１５６）,红细胞核的体积是鲫红细胞核体积的１．６倍。另一方面,用随机拉增多态ＤＮＡ（Ｒ     

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.     

Genetic and environmental variances on growth and reproductive traits of silver crucian carp Carassius langsdorfii using communal and separate rearing systems

SUMMARY: Five clonal lines of silver crucian carp Carassius langsdorfii were produced from natural maternal fish to estimate the variance and heritability of total length, bodyweight and gonadosomatic index (GSI). The larvae on the 10th day after hatching were reared together in a common rearing system with five duplicate systems. Another system was prepared using a separate system with two replications in each family. Estimation of variance and heritability were done in the first, third, fifth and 10th month for total length and bodyweight. Microsatellite DNA markers were used to identify the clonal lines in each communal rearing tank. Average heritabilities in the first month were 0.105 for total length (ranging from 0.21 to 0.03) and 0.127 for bodyweight (ranging from 0.22 to 0.06) then decreased in the third, fifth and 10th month. In contrast, intermediate levels of heritability from separate systems were observed. Likewise, relative heritability of GSI in the 10th month presented a high level in both rearing systems. Those heritabilities indicated that growth, total length, bodyweight and reproductive performances of silver crucian carp were affected by genetic influence. The results suggest that clonal lines of silver crucian carp are suitable as experimental animals for evaluations of environmental change and various pollutants through quantitative traits such as growth, reproductive and other physiological traits.     

Pharmacokinetics and tissue residues of marbofloxacin in crucian carp (Carassius auratus) after oral administration

Pharmacokinetics and residue elimination of marbofloxacin (MBF) were studied in crucian carp (Carassius auratus, 250±30 g) kept at two water temperatures of 15 and 25 °C. Marbofloxacin concentrations in plasma and tissues were analysed by means of high‐performance liquid chromatography using an ultraviolet detector. The limits of detection were 0.02 μg mL^?1, 0.02 μg g^?1, 0.025 μg g^?1, 0.02 μg g^?1 and 0.025 μg g^?1 in plasma and muscle, skin, liver and kidney respectively. Fish were administered orally at a single dosage of 10 mg kg^?1 body weight in the PK group. The data were fitted to two‐compartment open models at both temperatures. At 15 °C, the absorption half‐life () and distribution half‐life (t_1/2α) of the drug were 0.36 and 4.48 h respectively. The corresponding values at 25 °C were 0.23 and 0.87 h respectively. The elimination half‐life (t_1/2β) was 50.75 h at 15 °C and 25.05 h at 25 °C. The maximum MBF concentration (C_max) differed little between 15 (6.43 μg mL^?1) and 25 °C (8.36 μg mL^?1). The time to peak concentration was 1.74 h at 15 °C and 0.78 h at 25 °C. The apparent volume of distribution (V_d/F) of MBF was estimated to be 1.36 and 0.87 L kg^?1 at 15 and 25 °C respectively. The area under the concentration–time curve (AUC) was 301.80 μg mL^?1 h at 15 °C and 182.80 μg mL^?1 h at 25 °C. The total clearance of MBF was computed as 0.03 and 0.05 L h^?1 kg^?1 at 15 and 25 °C respectively. After repeated oral administration at a dosage of 10 mg kg^?1 body weight per day for 3 days, the results showed that the elimination half‐lives () of MBF from all tissues at 15 °C were longer than that at 25 °C. Therefore, water temperature is an important factor to be considered when deciding a reasonable withdrawal time.     

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.     

异育银鲫造血器官坏死症病鱼体内鲤疱疹病毒Ⅱ型的电镜观察与超微病理学特征

为分析患鲫造血器官坏死症异育银鲫Carassius auratus gibelio体内不同器官组织中鲤疱疹病毒(CyHV-2)粒子的形态结构、分布和发生过程,采集患病异育银鲫体肾、脾脏、头肾、肝胰脏、肠道、鳃各组织样本,利用透射电镜观察疱疹病毒和组织细胞超微病理学。在所采集器官组织中均观察到鲤疱疹病毒Ⅱ型的DNA内核、空衣壳、实心核衣壳、含包膜成熟病毒共4种不同成熟时期的病毒粒子,不同时期的病毒粒子直径分别为65~90nm、90~180nm、90~180nm、170~220nm;体肾和脾脏中含有大量的Cy HV-2病毒,而头肾、肝胰脏、肠道和鳃中病毒粒子较少;CyHV-2病毒主要感染体肾、头肾、脾脏的吞噬细胞,导致机体免疫机能改变;组织细胞病理学观察发现吞噬细胞核边缘化,核内染色质变性,核膜溶解,线粒体嵴断裂,出现空泡,个别细胞溶解坏死。通过PCR检测和电镜观察病毒粒子结构特征,确诊异育银鲫感染Cy HV-2病毒,主要器官组织细胞中均有CyHV-2病毒,且在细胞核中完成复制和组装,在细胞质中获得外膜。感染Cy HV-2病毒的异育银鲫主要器官组织均受到一定的损伤。     

Pharmacokinetics of minocycline in crucian carp (Carassius auratus) after intravenous and oral administration

The pharmacokinetic of minocycline was studied after a single intravenous as well as oral dose (5 mg/kg body weight) in crucian carp (Carassius auratus) reared in freshwater at 10°C. Plasma samples were randomly collected from six fish at each sampling time. Plasma concentrations were determined by high‐performance liquid chromatography and further subjected to noncompartmental analysis. Initial concentration of minocycline just after intravenous administration was calculated as 7.320 μg/ml, while the other parameters after intravenous injection were determined as flows: apparent elimination rate constant (λ_z) of 0.064 per hr, apparent elimination half‐life () of 10.82 hr, total body clearance (Cl) of 142.72 ml/hr/kg, volume of distribution (Vz) of 2,227.38 ml/kg and volume of distribution at steady‐state (Vss) of 1,937.08 ml/kg. While after oral administration, the λ_z, , mean absorption time (MAT), absorption half‐life (t_1/2ka) and bioavailability were determined as 0.059 per hr, 11.74, 5.55, 3.84 hr, and 81.98%, respectively, and the peak concentration was observed as 1.474 ± 0.362 μg/ml at 8 hr. It was shown that minocycline was slowly but relatively completely absorbed, extensively distributed, and slowly eliminated in crucian carp. Based on the ratios of AUC_0–24 hr/MIC90, a minocycline dosage of 5 mg/kg body weight administered intravenously or orally would be only effective to successfully treat crucian carp infected by bacterium with MIC values ≤0.25 μg/ml.     

饲料Vc对异育银鲫的生理效应及其适宜添加量

在精制饲料中分别添加不同剂量Vc(0、37.5、75、15 0、30 0、6 0 0和 12 0 0mg/kg) ,连续投喂异育银鲫 (方正银鲫♀×兴国红鲤♂ ) (Carassiusauratusgitelio) 8周。结果显示 ,当饲料中缺乏Vc时异育银鲫虽没有出现明显的缺乏症状 ,但日增重率、血红蛋白含量和血清POD酶活性均低于Vc添加组 ,而红细胞脆性极显著地高于高剂量Vc添加组 (P <0 .0 1) ;同时 ,经新复极差检验 ,在适宜范围内随着饲料Vc含量增加 ,异育银鲫日增重率、血红蛋白含量、POD活性等指标显著提高 ,而红细胞脆性明显下降 (P <0 .0 5 )。综合以上指标的非线性回归分析结果 ,建议生产上异育银鲫饲料Vc适宜添加量为 30 0～ 5 0 0mg/kg。     

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.     

聚六亚甲基胍对异育银鲫鳃出血病药效的初步研究

通过PCR检测及序列对比分析,确诊江苏海丰农场两塘口内异育银鲫(Carassius auratus gibelio)所患疾病为鲤疱疹病毒Ⅱ型(Cyprinid herpesvirusⅡ,Cy HV-2)引发的鲫造血器官坏死症(Crucian CarpHematopoietic Necrosis),而后分别每10 d使用0.5 m L/m~3和0.75 m L/m~3聚六亚甲基胍(Polyhexamethylene guanide,PHMG)泼洒治疗,并定期采样,利用Real Time PCR测定样品中病毒表达量。结果显示:该病毒(DF2015)Cy HV-2的DNA解旋酶基因序列长为316 bp,与绝大多数病毒株有较近的亲缘关系(99%),但与病毒株ST-J1和H.Fukuda的亲缘关系相对较远(63%)。与其余病毒株等有较近的亲缘关系(99%);治疗近2个月后,两塘的病毒相对表达量均呈下降趋势,且具有显著差异性,但0.75 m L/m~3治疗塘病毒相对表达量极显著降低,治疗效果相对更显著。     

彭泽鲫的分子遗传分析及其与方正银鲫A系的比较

Genetic homogeneity between Pengze crucian carp and strain A of silver crucian carp was studied by using transferrin,isozyme and RAPD markers.The studied individuals of Pengze crucian carp showed transferring patterns were the same of silver crucian carp A strain while distinct from those of other crucian carp populations.As far as isozyme is concerned,the MDH,LDH and EST are all of the same with only slight differences in SOD between them.The RPAD patterns clearly indicated high homogeneity among 16 individuals (6 sampled from individuals of two years old and the others aged one) from crucian carp of Pengze and 5 individuals from strain A of silver crucain carp.Nearly indentical banding patterns were observed among all individuals.Average genetic distance within all the individuals is only 0.011,suggesting crucian carp of Pengze might possess indentical genetic background with strain A of silver crucian carp.