Koi herpesvirus and carp edema virus threaten common carp aquaculture in Croatia

Common carp (Cyprinus carpio) is a very important fish species for warm-water aquaculture in Croatia. All Croatian carp farms are subjected to a surveillance programme for the presence of koi herpesvirus (KHV), causing a deadly disease called koi herpesvirus disease (KHVD). However, there is no surveillance for other viral pathogens of importance like carp edema virus (CEV), a causative agent of koi sleepy disease (KSD). During regular testing within the KHVD surveillance programme, we tested samples for CEV simultaneously. The screening indicated possible outbreaks of KHVD and KSD. During 2016, KHVD broke out in an isolated area and soon thereafter a KHV eradication programme was successfully performed. However, during 2018 and 2019, two additional mortality events occurred in lakes in the southern part of Croatia during the spring. Samples from both events tested positive for CEV. An epidemiological investigation confirmed the introduction of infected carps from an infected farm to one of the lakes. To prevent the spreading of CEV into open waters, it is of utmost importance to introduce CEV testing before fish movement or to perform regular testing of all carp farms in the country to determine CEV prevalence for the purpose of implementation of control measures.     

Whole-genome sequence of a novel Chinese cyprinid herpesvirus 3 isolate reveals the existence of a distinct European genotype in East Asia

Cyprinid herpesvirus 3 (CyHV3), also known as koi herpesvirus (KHV), can be subdivided primarily into European and Asian genotypes, which are represented by CyHV3-U or CyHV3-I and CyHV3-J, respectively. In this study, the whole genome sequence of a novel Chinese CyHV3 isolate (GZ11) was determined and annotated. CyHV3-GZ11 genome was found to contain 295,119 nucleotides with 52.9% G/C content, which is highly similar to those of published CyHV3-U, CyHV3-I, and CyHV3-J strains. With reference to CyHV3-U, CyHV3-I, and CyHV3-J, CyHV3-GZ11 was also classified into 164 open reading frames (ORF), which include eight repeated ORFs. On the basis of the 12 alloherpeviruses core genes, results from phylogenetic analysis showed that CyHV3-GZ11 had closer evolutionary relationships with CyHV3-U and CyHV3-I than with CyHV3/KHV-J, which were also supported by genome wide-based single nucleotide substitution analysis and the use of a series of developed molecular markers. This study was the first to reveal the presence of a distinct European CyHV3 genotype in East and Southeast Asia at a whole genome level, which will evoke new insights on exploring the origin, evolution, and epidemiology of the virus.     

瓯江彩鲤与日本锦鲤及其正反杂交F1形态特征和生长初步研究

对瓯江彩鲤、日本锦鲤及其正反杂交F1采用同塘试验比较生长率，采用聚类分析和判别分析比较分析形态差异。结果表明，正反杂交F1的瞬时增重率低于双亲，而绝对增重率介于双亲之间；正反杂交F1形态性状有的介于双亲之间，有的表现为超亲本优势，有的性状低于双亲的性状值。总体上，日本锦鲤对正反杂交F1的遗传影响明显大于瓯江彩鲤，2个亲本基因的相互作用在遗传过程中也起到了重要的作用。     

Physiologic and biochemical measurements and response to noxious stimulation at various concentrations of MS‐222 in Koi (Cyprinus carpio)

ObjectiveTo evaluate the physiological effect and response to noxious stimulation at five concentrations of MS-222 in koi (Cyprinus carpio).Study designProspective experimental study.AnimalsTwenty-one healthy adult unknown sex koi fish weighing mean 450 ± SD 120 g.MethodsEach fish was exposed to five different concentrations of MS-222 (50, 70, 110, 150 and 190 mg L^?1) in a random sequence during the same anaesthetic event. For each concentration of MS-222, vital functions such as heart rate (HR) (via Doppler) and opercular rate (OpR) were recorded after a standardized induction period. Response to two noxious stimuli in the form of haemostat clamp pressure applied on the tail and the lip was evaluated, and blood was drawn to measure biochemical and blood gas values.ResultsDecrease in response to noxious stimulation with an increase of MS-222 concentration both for the lip (p = 0.0027) and the tail (p < 0.0001) stimulus was observed. Biochemical values were unaffected by the concentration of MS-222 with the exception of lactate concentration which was weakly correlated with the duration of anaesthesia (r = 0.31, p < 0.001) and the number of times the fish was clamped or bled prior to sampling (r = 0.23, p < 0.001). Opercular rate decreased with the increase in anaesthetic concentration, and HR was not affected.Conclusions and clinical relevanceOur results indicated a decrease in response to stimulus and a decrease in OpR that were associated with increased concentrations of MS-222. This may assist in establishing anaesthetic protocols using MS-222 in fish and supports the use of supramaximal pressure stimuli to teleost fish under variable MS-222 concentrations as a model for future studies.     

一例冰下低温爆发的锦鲤疱疹病毒病的鉴定

2013年4月初低温下，吉林省白山地区某网箱养殖场鲤（Cyprinus carpioL．）大量死亡，病死鲤无明显临床病变。采用PCR扩增技术检测了这些病鱼，结果表明，该低温爆发的疾病为锦鲤疱疹病毒病（KHVD）。这种病常爆发在水温为18-28℃的春秋季，冰下爆发还未有报道。本次锦鲤疱疹病毒病的鉴定，对于锦鲤疱疹病毒感染鲤温度条件的变化和疾病的预防提供了参考。     

锦鲤疱疹病毒囊膜蛋白ORF59的克隆、分析及其主要B细胞表位区的原核表达

从感染锦鲤疱疹病毒（Koi herpesvirus,KHV）的锦鲤（Cyprinus carpiokoi）肾脏组织中提取DNA,通过PCR扩增了KHVORF59基因。该基因全长411bp,所编码的蛋白包含136个氨基酸,分子量14.3kDa,等电点（PI）6.91,有12个潜在的O糖基化位点。此研究克隆的KHVORF59基因第130位碱基由G突变为A,使其编码的第44位氨基酸由Ala突变为Thr。采用DNAStar程序,在综合分析二级结构柔性区、蛋白的亲水性、表面可能性和抗原性指数的基础上,预测了KHVORF59蛋白主要B细胞表位,并将其区段的编码序列与KHVORF59完整编码序列分别克隆入原核表达载体pET-32a（＋）,构建重组质粒pET32a-ORF59S和pET32a-ORF59C,转入大肠杆菌Rosetta菌株,IPTG诱导表达。SDS-PAGE及WesternBlot分析显示,pET32a-ORF59S可以高效表达,表达的截短KHVORF59蛋白主要以可溶性形式存在,采用HisBindResin填料,层析纯化了该截短蛋白。     

A comparison of growth, survival rate and number of marketable koi carp produced under different management regimes in earthen ponds and concrete tanks

To compare the growth performance of koi carp, Cyprinus carpio var. koi, produced in concrete tanks (2.13 × 0.91 × 1.22 m; capacity: 2,000 l each) and earthen ponds (9.1 × 6.10 × 1.07 m; capacity: 59650 l each), fish larvae (stocking size: 0.12 ± 0.008 g) were cultured for 11 weeks and individual weight gain, survival rate and number of marketable fish produced were compared among four management regimes for each culture system: (1) live zooplankton fed to fish larvae in ponds (PLF) and tanks (TLF); (2) application of poultry manure in ponds (PPM) and tanks (TPM); (3) application of cow manure in ponds (PCD) and tanks (TCD); and (4) a control treatment for ponds (PC) and tanks (TC), where a commercial feed was applied. There were three replicates for each treatment. Weight gain of koi carp was highest in the PLF treatment, followed in decreasing order by TLF, PPM, PCD, TPM, TCD, PC and TC treatments (P < 0.05). There was a significant difference in the survival of koi carp among the treatments, ranging from 67.83% in TC to 95.50% in PLF. The number of marketable fish produced was highest in the PLF treatment, followed in decreasing order by TLF, PPM and PCD treatments. However, none of the fish produced in the TPM, TCD, PC and TC treatments attained marketable size. Significantly higher (P < 0.05) values of pH and dissolved oxygen (for water samples collected weekly at 9 A.M.) were obtained in the live food and control treatments (for both tanks and ponds), compared to the manured treatments. The concentration of total alkalinity, BOD, PO₄-P, NO₃-N and specific conductivity were significantly higher (P < 0.05) in PPM and PCD, compared to other treatments. NO₂-N and NH₄-N values were significantly higher (P < 0.05) in TPM and TCD, than other treatments. The results suggest that introduction of live zooplankton into culture units result in higher growth of koi carp larvae compared to manure based systems. Earthen ponds appeared to be better alternative to concrete tanks for manure application through maintenance of better water quality due to their higher assimilatory capacity and greater abundance of plankton which resulted in better growth of cultured fish.     

锦鲤摩氏摩根氏菌的分离鉴定与药敏性研究

为鉴定2009年山东某养殖场锦鲤病病原,本研究从病鱼体内分离到3株优势菌,经回归感染实验证实编号为A400502的分离株为该病的致病菌.该菌为革兰氏阴性杆菌,周生鞭毛,大小为0.5 μm～0.7 μm×1.0 μm～1.5 μm.其生化特征为鸟氨酸脱羧酶、尿素酶、酚红反应阳性,赖氨酸脱羧酶和精氨酸双水解酶阴性.利用16S rDNA序列分析和比对结果构建的系统发育树表明,该菌与登录号为EF455493的摩氏摩根氏菌(Morganella morganii)的同源性高达99％,因此将该菌鉴定为摩氏摩根氏菌.26种药物的敏感实验结果表明,该分离株对复达欣、庆大霉素、新霉素等11种药物高度敏感.     

锦鲤疱疹病毒-CJ株ORF136基因的原核表达及免疫原性研究

为了研究能有效预防锦鲤疱疹病毒的疫苗,本文研究克隆了锦鲤疱疹病毒ORF136基因,并构建重组质粒PET32a-ORF136,用IPTG诱导表达,SDS-PAGE检测获得含his标签大小为36.4kDa的蛋白条带。蛋白纯化后免疫鲤鱼,不同天数采血收集血清用间接ELASA检测抗体水平。结果显示：用10ug/尾、20ug/尾和40ug/尾的蛋白免疫后均可检测到KHV的抗体,用PET32a-ORF136表达的蛋白20ug/尾和40ug/尾的蛋白免疫后的抗体水平比10ug/尾剂量蛋白免疫后的抗体水平高但差异不显著（p＞0.05）。