Koi herpesvirus and carp oedema virus: Infections and coinfections during mortality events of wild common carp in the United States

Koi herpesvirus (KHV; cyprinid herpesvirus‐3) and carp oedema virus (CEV) are important viruses of common and koi carp (Cyprinus carpio); however, the distribution of these viruses in wild common carp in North America is largely unknown. During the summers of 2017 and 2018, 27 mass mortalities of common carp were reported from four states in the USA (Minnesota, Iowa, Pennsylvania and Wisconsin), the majority of which were distributed across eight major watersheds in southern Minnesota. Samples from 22 of these mortality events and from five clinically healthy nearby carp populations were screened for KHV, CEV and SVCV using real‐time polymerase chain reaction (qPCR). KHV was confirmed in 13 mortality events, CEV in two mortality events and coinfections of KHV/CEV in four mortality events. Nucleotide sequence analysis revealed that the KHV and CEV detected here are closely related to European lineages of these viruses. While molecular detection alone cannot conclusively link either virus with disease, the cases described here expand the known range of two important viruses. This is also the first reported detection of KHV and CEV coinfections in wild carp populations.     

First year growth and survival of common carp in two glacial lakes

Abstract  Cohorts of common carp, Cyprinus carpio Linnaeus, were monitored from hatch through the next spring in two South Dakota, USA lakes to identify factors affecting year-class strength. Hatching occurred over 37- and 47-day periods in the two lakes. Common carp that hatched earlier achieved greater total lengths at the end of the first growing season (Brant Lake: r  = −0.84, d.f. = 77, P  <   0.001; Campbell Lake: r  = −0.87, d.f. = 78, P  <   0.001). Mean daily growth rates were approximately 1.0 mm day⁻¹ and similar between lakes. Total length was positively related to daily growth rate in the two lakes, indicating that members of the cohort that grew faster achieved greater total lengths by time of capture. Hatch date was inversely related to daily growth (fish hatched earlier grew faster) in one lake ( r  = −0.44, d.f. = 77, P  < 0.001) but not the other ( r  = −0.04, d.f. = 78, P  = 0.678), suggesting that length at the end of the growing season can be affected by length of growing season and growth rate. Overwinter survival of age-0 fish appeared to be size dependent because age-1 common carp length–frequency distributions from both lakes comprised only of the larger cohort members present the previous autumn.     

鲤浮肿病毒人工感染锦鲤的试验研究

为研究鲤浮肿病毒的感染方式及在锦鲤各组织器官中的动态分布,在水温20～22℃,溶解氧7～8mg/L试验条件下,采用注射、浸泡、划伤后浸泡3种方式人工感染锦鲤,观察了试验鱼的发病和死亡情况;检测了鲤浮肿病毒在感染鱼体内各组织器官中的存在情况,探讨了主要被感染组织的病理变化。试验结果显示,采用注射、浸泡、划伤后浸泡3种方式均可导致健康锦鲤感染该病毒,病鱼出现昏睡、烂鳃、凹眼等症状,累积死亡率35%～88%。锦鲤感染该病毒后至少有3d的潜伏期,随后病毒在鳃组织中大量增殖,至7d达到高峰,病毒量314.8个/ng,之后病毒量开始逐渐下降,至12d在鳃组织中检测不出病毒。鲤浮肿病毒在肾、肝、脑、肌肉和脾中存在的时间较短且病毒量较低,在血液和肠道中未检出。结合鳃组织病理切片可知,鳃为该病毒感染的主要靶器官。本研究结果为鲤浮肿病的诊断和防控提供了一定的科学依据。     

鲤疱疹病毒2型和3型三重PCR检测方法的建立与应用

鲤疱疹病毒2型和3型主要感染鲫(金)鱼(Carassius auratus)、鲤鱼(Cyprinus carpio)和锦鲤(Cyprinus carpio haematopterus)及其杂交种,具有高度的传染性和致病性,对养殖鲤科鱼类危害严重。为了建立快捷、高效且能同时检测这2种类型鲤疱疹病毒的检测技术,本研究根据鲤疱疹病毒的DNA聚合酶基因(2、3型)、解旋酶基因(2型)和胸苷激酶基因(3型)的保守序列,设计了3对特异性引物,通过对三重PCR的反应条件进行优化,建立起鲤疱疹病毒2型和3型三重PCR检测方法,并运用该方法对实验室保存的鲫鱼和鲤鱼组织样品进行检测。结果显示,该三重PCR检测方法仅在鲤疱疹病毒阳性样品中扩增出3条特异性条带,表现出良好的特异性;以克隆的目的基因质粒为模板,进行10倍梯度稀释,该检测方法能检出的极限值为2.85×102 copies/μL,表现出较高的灵敏性;运用该方法检测122份鲫鱼和60份鲤鱼样品,其结果与运用国家标准(GB/T 36194-2018)和行业标准(SC/T 7212.1-2011)方法检测的结果一致。本研究表明,构建的三重PCR检测方法不仅具有较高的准确性和灵敏度,还能同时检测2种类型的鲤疱疹病毒,有效提高检测效率。     

Carp edema virus in Polish aquaculture – evidence of significant sequence divergence and a new lineage in common carp Cyprinus carpio (L.)

Fish samples initially collected by local veterinarians on the common and koi carp farms in Poland between 2013 and 2015 as part of a KHV surveillance programme, when the water temperature was between 16 and 26 °C, and were also tested for CEV by qPCR. A partial 478 nucleotide fragment of the 4a gene was subsequently generated from 17 qPCR‐positive common carp Cyprinus carpio samples from 36 farm sites tested during the period. Sequence alignments and analysis revealed the presence of CEV in Poland both in common carp as well as in koi carp farms, and phylogenetic analysis assigned the Polish CEV sequences into three distinct genogroups. A lineage which includes the original sequences obtained from koi carp in Japan (genogroup II) included sequences from both koi carp and common carp, and the second lineage (genogroup I) contained sequences from common carp only. A third lineage (genogroup III) which was more closely related to the genogroup II also consisted of sequences from common carp only. The latter represents a lineage of CEV not previously described in the literature.     

Effect of d-Lys6 salmon sGnRH alone and in combination with domperidone on the spawning of common carp during the late spawning season

The effect of [d-Lys6] salmon gonadotropin-releasing hormone alone (sGnRH) and in combination with domperidone, a dopamine antagonist, on the spawning of common carp, Cyprinus carpio, was investigated during the late spawning season (when mature fish stop spawning under natural conditions when males and females are put together). Fish were induced to spawn by [d-Lys6]-sGnRH salmon gonadotropin-releasing hormone analogue (sGnRH-X) only when injected in combination with domperidone at doses of 10 g kg-1 and 20 mg kg-1 body weight respectively. [d-Lys6]-sGnRH-X alone did not induce spawning at a dose as high as 100 g kg-1 body weight. Fish injected with domperidone alone did not spawn either. These results demonstrate that [d-Lys6]-sGnRH-X in combination with a dopamine antagonist is effective in inducing spawning during the late spawning season.     

Carp edema virus from three genogroups is present in common carp in Hungary

Hungary is an important carp producer with intensive trading relationships with farms in other carp‐producing areas in Europe. Carp in Europe were recently found infected with carp edema virus (CEV), a poxvirus which causes the koi sleepy disease (KSD) syndrome. Moribund carp were collected from 17 fish farms and angling ponds in different regions of Hungary. Histological analysis of gills from these carp revealed a proliferation of the interlamellar epithelium and an infiltration by eosinophilic cells. In 13 of 17 of these carp, CEV DNA was detected by qPCR and in seven fish more than 1 × 10⁴ copies of virus‐specific DNA sequences per 250 ng of DNA, which could be considered as clinically relevant and a cause of disease. A phylogenetic analysis of the sequences revealed that all three genogroups of CEV were present in Hungarian common carp with genogroup I being most abundant. These results support the hypothesis of a prolonged presence of CEV in European carp populations and suggest that previous outbreaks of KSD were not recorded or misdiagnosed. Hence, a testing of carp and koi for infection with CEV should be included into disease surveillance programmes to prevent further spreading of this disease.     

鲤疱疹病毒2型武汉株的分离与鉴定

采用细胞培养、超薄切片电镜观察、巢式PCR检测以及病毒DNA克隆与测序分析等技术, 从湖北武汉一循环水养殖系统中患出血病异育银鲫(Carassius auratusgibelio)体内分离鉴定了一株鲤疱疹病毒2型(Cyprinid herpesvirus 2, CyHV-2)病毒, 命名为鲤疱疹病毒2型武汉株(CyHV-2-WH)。患病鱼组织匀浆液接种锦鲤鳍条细胞系(Koi-Fin)可产生典型的细胞病变效应。用患病鱼组织匀浆液与细胞培养物分别人工感染异育银鲫, 均可重复出与自然发病相同的症状, 死亡率达100%。患病鱼脾与肾脏组织超薄切片电镜观察结果显示, 病毒为具囊膜的球形病毒, 囊膜直径为170~200 nm, 病毒衣壳直径为110~120 nm, 病毒在细胞核内复制、组装。采用CyHV-2的特异引物对病鱼样本和感染细胞培养物样本进行巢式PCR检测, 均能扩增出357 bp的目的条带, 将该扩增产物进行测序与比对分析后发现, 其与GenBank中已报道的CyHV-2毒株的DNA解旋酶基因高度同源(99.44%以上), 与鲤疱疹病毒3型(CyHV-3)也有较高的同源性(81.55%)。系统进化分析结果显示, CyHV-2-WH株与JS2012、H.Fukuda、YC110907等CyHV-2病毒株属同一分支。

     

First feeding of common carp larvae on diets with high levels of protein hydrolysates

A 21 day feeding trial was carried out at 24 °C aiming to evaluate the effect of diets containing high levels of protein hydrolysates on growth, survival and body composition of common carp, Cyprinus carpio L., larvae since first feeding. Eight semipurified diets based on a fish protein hydrolysate (CPSP) with or without additional nitrogen sources, such as casein, casein hydrolysates or other hydrolysates from several origins, were tested, using a diet based on yeast as a reference. High survival rates of larvae were observed at the end of the trial for all experimental diets, ranging from 97% for the yeast diet to 71% when CPSP was used as the only nitrogen source. In terms of growth, the best results were achieved with the CPSP-casein diet (1:1) and the yeast-based diet (17.5 mm total length / 82.8 mg wet weight and 16.3 mm total length / 55.4 mg wet weight, respectively). Growth of larvae fed on diets with protein hydrolysates as the only nitrogen source was considerably lower than that of larvae fed a mixture of intact protein and protein hydrolysates.     

Influence of management protocols on carp growth under nursery conditions: relative importance of food and water quality

Twenty outdoor holding tanks (10 m–3 each), were filled with ground water and grouped into five feeding schemes: live zooplankton cultured outside the fish growing tank (LFS); direct nursery pond fertilization schedule in static (MS) as well as in exchanged water (EMS); intermediate conditions between the LFS and MS (IS); and supplementary food system using mixture (1:1) of finely ground mustard oilcake and boiled rice (SFS). Two hundred common carp (Cyprinus carpio) fry were introduced into each of the twenty outdoor tanks maintained at ambient temperature outside the laboratory. After 90 days of rearing, the fish were harvested and analysed for various growth parameters and food conversion efficiency. The average weight of carp attained in the LFS was significantly higher than that in the other four feeding schemes. The frequency distribution of final body weight of common carp showed the preponderance of large and small fish in the LFS and MS, respectively. Likewise, plankton intake by the carp fry was highest in the LFS. The rate of survival was much higher in the LFS as compared with the rest of the treatments. The water quality remained far better in the former than the latter. © Rapid Science Ltd. 1998     

Inactivation of koi‐herpesvirus in water using bacteria isolated from carp intestines and carp habitats

Since its first outbreak in Japan in 2003, koi‐herpesvirus (KHV) remains a challenge to the carp Cyprinus carpio L. breeding industry. In this study, inactivation of KHV in water from carp habitats (carp habitat water) was investigated with the aim of developing a model for rapidly inactivating the pathogen in aquaculture effluent. Experiments with live fish showed that, in carp habitat water, KHV lost its infectivity within 3 days. Indications were that inactivation of KHV was caused by the antagonistic activity of bacteria (anti‐KHV bacteria) in the water from carp habitats. Carp habitat water and the intestinal contents of carp were therefore screened for anti‐KHV bacteria. Of 581 bacterial isolates, 23 showed anti‐KHV activity. An effluent treatment model for the disinfection of KHV in aquaculture effluent water using anti‐KHV bacteria was developed and evaluated. The model showed a decrease in cumulative mortality and in the number of KHV genome copies in kidney tissue of fish injected with treated effluent compared with a positive control. It is thought that anti‐KHV bacteria isolated from the intestinal contents of carp and from carp habitat water can be used to control KHV outbreaks.     

Detection of Koi herpesvirus: impact of extraction method,primer set and DNA polymerase on the sensitivity of polymerase chain reaction examinations

Since virus isolation is seldom successful, KHV infection is commonly detected by PCR examination. A number of different PCR assays have been described in recent years. However, at present no commonly accepted PCR method is used amongst different laboratories. The aim of this study was to check if the examination of infected fish by different PCR methods yielded comparable results. We used tissue samples of three KHV‐infected koi, one KHV‐infected common carp, one KHV‐infected goldfish and one non‐infected common carp. DNA was extracted with DNAzol Reagent, High Pure PCR Template DNA Preparation Kit and QIAamp DNA Mini Kit. The DNA was tested by PCR with different combinations of published primer sets –KHV‐F and ‐R, KHV‐Gray‐2F and ‐2R and KHV‐TKf and ‐TKr – plus different DNA polymerases – a standard Taq DNA polymerase, a Platinum (hotstart) Taq DNA polymerase and a Platinum (hotstart) Pfx DNA polymerase with proofreading activity. The different extraction methods produced DNA solutions with different yields of DNA and different degrees of homogeneity. Also, the sensitivity of the PCR depended on the choice of the primer set and polymerase. Not all infected fish could be identified with all methods; there were large differences in the sensitivity between methods.     

Koi herpesvirus epizootic in cultured carp and koi, Cyprinus carpio L., in Taiwan

Koi herpesvirus (KHV) poses a significant threat to cultured koi and common carp, both Cyprinus carpio L. Since the first reported case in Israel in 1998, KHV has rapidly spread worldwide. This study investigates the spread of KHV to Taiwan by collecting 49 cases of suspected common carp and koi infections from 2003 to 2005 for analysis. Clinical signs included lethargy, anorexia, increased respiratory movements and uncoordinated swimming. Hyperaemia, haemorrhage on body surface and necrotic gill filaments were recorded. Gill epithelial hyperplasia, necrosis and eosinophilic intranuclear inclusion bodies were observed by histological examination, while virions were detected using transmission electron microscopy. By detecting the presence of the KHV thymidine kinase (TK) gene and the KHV 9/5 gene using polymerase chain reaction (PCR), 37 cases were identified as KHV-positive, and the cumulative mortality of infected fish was 70-100%. Positive cases showed identical sequences for the genes analysed, implying that they were of the same origin. For the KHV 9/5 gene sequence, these cases exhibited 100% identity with the Japanese strain (TUMST1, accession number AP008984) and 99% identity with the Israeli (KHV-I, DQ177346) and US (KHV-U, DQ657948) strains. Additionally, a loop-mediated isothermal amplification (LAMP) assay was performed and found to be more sensitive than PCR tests, suggesting its potential use as a rapid diagnostic method for KHV. This is the first epidemiological study of KHV infection in cultured common carp and koi in Taiwan.     

Antibody response of two populations of common carp, Cyprinus carpio L., exposed to koi herpesvirus

Common carp, Cyprinus carpio L., exposed to koi herpesvirus (KHV) may become persistently infected and populations containing such virus-infected individuals may transmit the virus to other fish when co-habited. Detection of virus-infected fish in a population is thus critical to surveillance and control programmes for KHV. A study was therefore designed to detect anti-KHV serum antibodies, with an enzyme-linked immunosorbent assay, in common carp following experimental exposures to KHV under varying environmental conditions. The study determined that a proportion of fish within a population experimentally exposed to KHV (at least 10–25%) develop high antibody titres (1/1600 or greater) to the virus, and this immunological response was detectable for several months (observed at the termination of the experiments at 65, 46 and 27 weeks post-exposure). Furthermore, this response was detected in one population of fish that did not succumb to a high level of mortality when maintained at water temperatures that were non-permissive for KHV. Elevating the water temperatures to permissive conditions for KHV resulted in recurrence of disease despite the presence of anti-virus antibodies, suggesting that serum antibodies alone are not protective under the conditions of our trials.     

Anoxia survival in common carp and crucian carp is related to high zinc concentration in tissues

ABSTRACT:   Ten lots of common carp and six lots of crucian carp (each lot of 100 fish) were treated under anoxia for five days at water temperatures of 25–31°C. The average per group mortality of common carp was 17%, but none of the 600 crucian carp died. The digestive tract tissues of the common carp that died had significantly lower zinc concentrations than those that survived (59 ± 41 vs 142 ± 60 μg/[g fresh tissue], P  < 0.001). The digestive tract tissues of the crucian carp had mean zinc concentrations of 652 ± 458 μg/(g fresh tissue). One lot of common carp that had low tolerance for anoxia was fed a high zinc diet (2000 mg zinc/kg diet) for 1, 2 or 6 months and then subjected to 5 days anoxia. The survival rates of those fed the high zinc diet 1 and 2 months increased from 0 to 50%, respectively; all of fish that had fed a high zinc diet for 6 months survived. Thus, anoxia survival in common carp and crucian carp is closely related to the high concentrations of zinc in their tissues.     

Effects of dietary medium-chain triacylglycerols (tricaprylin and tricaproin) and phospholipid supply on survival, growth and lipid metabolism in common carp (Cyprinus carpio L.) larvae

The present study investigated the interaction of dietary medium-chain fatty acids (MCFA) and phospholipids (PL) on survival, growth and lipid metabolism in common carp larvae. Nine diets based on casein and dextrin and with a variable lipid part were tested in triplicate for 22 days post first feeding. The 3×3 design consisted of three triacylglycerols (3% of diet) combined with three different lipid supplements. Tested triacylglycerols were triolein (TOL), tricaprylin (TC8) and tricaproin (TC6), and lipid supplements were 2% soybean oil (low-fat diets without PL), 2% soybean lecithin (low-fat diets with 2% PL) or both 2% soybean lecithin and 6% TOL (high-fat diets with 2% PL).

In the first step, both TC6 and TC8 resulted in improved survival and growth rates compared to TOL, irrespective of the PL supply. In the second step, TC8 decreased survival and growth rates, whereas the difference between TC6 and TOL became less. Histological signs of impaired intestinal absorption of neutral lipids were evidenced in larvae fed TOL without PL and also in high-fat diets with 2% PL. The latter diets also resulted in poorer growth rates compared to low-fat diets with 2% PL. These results suggest that the quantitative PL requirement of larvae increases as the dietary level of long-chain triacylglycerols increases. Larvae fed TC6 or TC8 showed enlarged liver and hepatocyte volume and a decreased level of body neutral lipids. Based on β-hydroxybutyrate (β-HBA) measurements in whole larvae, TC8 was found to be more ketogenic than TC6. TC6 and TC8 affected differently the fatty acid profile of larval body neutral lipids. TC6 did not induce the appearance of MCFA, whereas TC8 feeding resulted in a low level of 8:0 and relatively high levels of 10:0 (3.8% of total fatty acids). Neither 8:0 nor 10:0 were found in larval polar lipids.

This study confirmed the essentiality of PL in common carp larval diets and underlines differences in the utilization of TC6 and TC8, which both initially stimulate growth during the first week, but only temporarily in the case of TC8.     

鲤胰蛋白酶原基因的电子克隆及生物信息学分析

为深入研究胰蛋白酶在鱼类中的生理功能和作用机制,利用生物信息学的方法,成功获得了鲤3种胰凝乳蛋白酶原cDNA序列(ccTRP1、ccTRP2和ccTRP3)并对其进行了序列分析。结果表明,三者均含有一个长度为729bp的开放阅读框(open reading frame,ORF),编码由242个氨基酸组成的胰蛋白酶原,其中包括15个氨基酸组成的信号肽和5个氨基酸(LDDDK)组成的激活肽。总平均亲水性GRAVY(grand average of hydropathicity)分析表明,三者均是亲水性蛋白。氨基酸比对结果显示,三者具备胰蛋白酶原的保守结构特征,如含有催化三联体氨基酸(His-57、Asp-102和Ser-195),12个半胱氨酸,位于底物结合口袋底部Asp-189和口袋开口处的Gly-216、Gly-226等。同时,鲤3种胰蛋白酶之间具有90%以上的同源性。进化树结果显示,鲤3种胰蛋白酶均属于Group I(阴离子型胰蛋白酶),且三者的进化距离不一致。     

Rapid detection and differentiation of carp oedema virus and cyprinid herpes virus‐3 in koi and common carp

Carp oedema virus (CEV) and koi herpes virus (KHV) are of major concern to common carp breeders and koi enthusiasts worldwide. The viruses cause diseases that exhibit similar external signs; thus, it is difficult to distinguish between them clinically. In this study, we developed and optimized rapid and accurate single‐ and multiplex isothermal diagnostic tools, based on recombinase polymerase amplification (RPA), for detection and differentiation of CEV and KHV. The assays were combined with a lateral flow dipstick to enable visual detection of amplification products and simplify post‐amplification analysis. Both CEV‐ and KHV‐RPA assays were specific for their target virus. The lower detection limits of the assays were similar to those of established diagnostic PCR tests for the viruses. A sample preparation method was optimized to eliminate the need for total DNA extraction from fish tissues. The estimated time to perform these RPA assays, from receiving the sample to having a result, is 50 min, compared to 10 and 7 hr for CEV‐ and KHV‐PCR tests, respectively. The assays can be performed in field situations to improve screening of fish and reduce spread of these viruses and thereby enhance the common carp and koi industries.     

水晶彩鲫、红鲫和锦鲤的腹膜脏层黑色素观察

对水晶彩鲫(Carassius auratus transparent coloredvar.)、红鲫(C.a.redvar.)和锦鲤(Cyprinus carpio ornamentalvar.)腹膜脏层黑色素进行肉眼观察和密度分布统计。结果表明,这3种鱼的不同体色个体,腹膜脏层黑色素的密度不同。依据黑色素的密度,将其分为Ⅰ~Ⅵ级。水晶彩鲫的腹膜脏层黑色素密度:肉白a个体为Ⅰ级(无黑色素分布),肉白b和红白个体多数在Ⅰ~Ⅱ级,红色个体多数在Ⅰ~Ⅳ级,杂色个体多数在Ⅳ~Ⅵ级,密度表现出依次递增的趋势。红鲫的腹膜脏层黑色素密度:白色个体分布在Ⅲ级,红白个体分布在Ⅲ~Ⅳ级,红色个体分布在Ⅲ~Ⅴ级,杂色分布在Ⅳ~Ⅵ级,青灰色分布在Ⅴ~Ⅵ级,密度依次也表现出递增的趋势。锦鲤的腹膜脏层黑色素密度:体表没有黑色斑纹的红、黄、白、红白和黄白等个体为Ⅰ级,无黑色素分布;体表有黑色斑纹的红黑、黄黑、白黑和红白黑等个体多数有黑色素出现,主要分布在Ⅳ级。[中国水产科学,2007,14(1):144-148]