锦鲤疱疹病毒GZ1301株的分离与鉴定

2013年4月,广东省一锦鲤养殖场暴发不明病因疾病,濒死锦鲤在塘边游动缓慢直至死亡,死亡率高达100%。现场采样发现,发病锦鲤体长25 cm,眼球凹陷,胸鳍及腹鳍出现出血斑点,解剖发现内脏器官包括肝、脾、肾肿大。细菌分离结果显示,内脏器官肝脏和肾脏中未分离到细菌。提取自然发病鱼的肝、脾、肾、鳃组织DNA作为模板,采用世界动物卫生组织(OIE)推荐的锦鲤疱疹病毒(KHV)检测引物进行PCR扩增,均能扩增出预期大小的特异性产物。NCBI的Blast搜索结果显示,扩增序列与KHV胸苷激酶(thymidine kinase,TK)基因核苷酸序列同源性为99%。病鱼内脏组织研磨过滤除菌后,腹腔注射20尾锦鲤,可复制出与自然发病相似的症状,并于7 d内全部死亡。取病鱼的鳃和肾脏研磨过滤除菌后进行细胞感染实验,结果显示,组织滤液感染CCB细胞后,盲传5代可以观察到典型的细胞病变效应(CPE)。将出现典型CPE的CCB细胞进行超薄切片制备和电镜观察,电镜下病毒呈对称20面体,直径约100 nm。将出现典型CPE的细胞进行间接免疫荧光实验,可以观察到特异性荧光。根据TK基因全长序列建立系统进化树,证实该毒株为KHV亚洲型毒株,暂命名为KHV-GZ1301株。研究结果可为KHV起源进化、分类以及疾病防控提供重要材料。     

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.     

Feed technology and production performance of Thai koi (Anabas testudeneus) in Bangladesh

ABSTRACT

Thai koi farming is relatively new in the aquaculture industry of Bangladesh. Costly industrial feed pellets is the main factor influencing the productivity of Thai koi (Anabas testudineus). Two types of feed, namely, floating and sinking, are commercially available in the country. The ingredients of both types of feed are more or less the same. Floating feed requires high technology, high pressure and temperature and hence is more expensive than sinking feed. Most of the Thai koi farmers use either floating or sinking feed, while some farmers use a mix of both types of feed. This article investigated the effects of these three feed types on Thai koi productivity using primary data collected from the Mymensingh district of Bangladesh. The performance of different feed types on productivity was investigated using the Cobb-Douglas production function and propensity score matching (PSM) methods. Floating feed led to the highest daily mean weight gain of fish, feed conversion ratios, and net returns. The regression and PSM results revealed that farmers using floating feed produced significantly higher quantity of fish per unit compared to those using the other feed types. The cost of feed per hectare was higher in the farms using the floating feed, but these farms earned extra return from the additional investment, compared with the farms that use the other feed types. Thai koi farmers are advised to use floating feed to reap higher production and returns on investment.     

锦鲤疱疹病毒天津株的分离与鉴定

为分析近年来天津地区养殖鲤(包括锦鲤)暴发性死亡的病因,利用病原菌分离、细胞培养、电镜观察、组织病理切片、人工感染实验、PCR和荧光定量PCR检测、基因分型等方法对患病鱼及病原进行了研究。结果显示,在患病鱼体表未发现大量寄生虫;在肝、脾、肾等内脏组织中未能分离到细菌;在鳃组织中发现大量的圆形病毒颗粒;使用患病鱼组织滤液感染锦鲤鳍条原代细胞,可观察到典型的细胞病变效应(CPE),注射患病鱼组织滤液和产生病变的细胞上清液可分别导致健康锦鲤93.3%和86.7%的死亡率;通过病理组织切片观察,主要病变组织为鳃、肝脏和肾脏。采用世界动物卫生组织(OIE)推荐的锦鲤疱疹病毒(KHV)检测方法进行PCR检测发现KHV呈阳性,且KHV在鳃组织中含量最高,肾脏次之,脑组织中最少。结合TK基因全长序列建立系统进化树和基因型分析,证实此次分离到的KHV为亚洲型毒株,属于I++II–基因型,将其命名为KHV-TJ1601株。这是我国华北地区首次报道KHV I++II–基因型的存在,可为KHV的进化分析和疫苗制备提供基础资料。     

Verification of mitotic gynogenesis in ornamental (koi) carp (Cyprinus carpio L.) using microsatellite DNA markers

The Japanese ornamental (koi) carp is a popular decorative fish all over the world. In koi, clones have not yet been obtained, although production of fish with identical colour patterns could be of commercial interest. Mitotic gynogenetic progenies are essential for subsequent production of clones in fish. However, resulting late‐shocked progenies may be contaminated with meiotic gynogens from spontaneous suppression of the second meiotic division in eggs. In this study, microsatellite DNA markers were used to confirm mitotic gynogenetic origin of obtained late‐shocked progenies. Recombination rate (y) and mapping distance relative to centromere (M‐C) of 10 microsatellite loci were determined based on percentage of heterozygotes in meiotic gynogenetic progenies. The range of y varied from 0.01 to 0.96 and the M‐C map ranged from 0.5 to 48 cM. The mean value of y over the 10 loci was 0.481. Six loci, which had y 0.47 and higher, were used as markers in two late‐shocked gynogenetic progenies. Complete homozygosity was revealed at all six microsatellite loci indicating mitotic gynogenetic origin of analysed progenies.     

Isolation and characterization of koi herpesvirus (KHV) from Indonesia: identification of a new genetic lineage

Koi herpesvirus (KHV) is the aetiological agent of an emerging disease (KHVD) associated with mass mortalities in koi and common carp and reported from at least 30 countries. We report the first isolation of KHV from koi and common carp in Indonesia and initial characterization of the isolates. Clinical signs, histopathology and virion morphology are similar to those of isolates from other countries. Phylogenetic analyses using the thymidine kinase gene amplified from each isolate and from carp tissue samples collected from KHVD outbreaks throughout Indonesia indicated that the Indonesian isolates are more closely related to the Asian than the European KHV lineage. Sequence analysis of two other variable regions between ORF29 and ORF31 (marker I) and near the start of ORF 133 (marker II) indicated that all Indonesian isolates displayed a marker I allele (I(++)) previously identified only in isolates of the Asian lineage. However, in the marker II region, all Indonesian isolates displayed the II(-) allele, which has been reported previously only amongst isolates of the European lineage, and nine of these displayed a mixed genotype (II(+)II(-)). The I(++)II(-) genotype has not been reported previously and appears to represent a new intermediate lineage that may have emerged in Indonesia.     

池塘精养日本锦鲤成鱼试验

日本锦鲤是一种集观赏、垂钓、美食于一体的较大型鱼类 ,素有“水中瑰宝”之美誉。其体色艳丽、游姿潇洒、斑纹华丽 ,且易于饲养 ,越来越受人们的青睐。 2 0 0 1年 3月 16日至 11月 10日我们进行了日本锦鲤成鱼的池塘集约化养殖试验 ,取得较好的效益 ,现将试验总结如下。1　材料与方法1 1　池塘条件 :试验池为洛阳市郊区面积为 2 5亩的长方形池塘。池埂为砖混结构 ,池深为 2m以上 (可保持水位 1 5m以上 ) ,水源采用无污染的地下水 ,且注水、排水方便。池中配备 1 5的叶轮式增氧机一台。1 2　鱼种放养 :2 0 0 1年 3月 16日从洛阳市水产科…     

Transformation of mugwort (Artemisia vulgaris) oil into nanoemulsion as an ethanol-free herbal anesthetic for surgery on koi carp (Cyprinus carpio)

Fish are frequently anesthetized to reduce stress and injury during various procedures, particularly surgery. Nonetheless, the majority of chemical fish anesthetics have some negative effects on both fish and humans. 2-Phenoxyethanol (2-PE), for example, which is normally used as an anesthetic for ornamental fish, can cause neuropsychological syndromes in humans. To reduce toxicity, some herbal essential oils have been applied as an anesthetic to fish. However, essential oils must be dissolved in ethanol, which has side effects such as disrupting gas exchange in fish. This study aimed to develop mugwort (Artemisia vulgaris) oil as a nanoemulsion without ethanol and determine their optimal concentrations to use as an anesthetic for surgery on koi carp (Cyprinus carpio). Gas chromatography-mass spectrometry (GC/MS) was used to examine the chemical compositions of mugwort oil. The droplet size of mugwort oil nanoemulsion was measured by the dynamic light scattering method. The results revealed that mugwort oil consisted of three major active compounds which were b-pinene (11.7%), 1,8-cineole (10.34%), and a-pinene (8.29%). The average diameter of mugwort oil nanoemulsion droplets was 22.08 ± 0.70 nm. The lowest effective concentrations were 0.6 ml/L for mugwort oil and 12.5 ml/L for mugwort oil nanoemulsion.

     

Stochastic modeling of production risk and technical efficiency of Thai koi (Anabas testudineus) farming in Northern Bangladesh

Thai koi is one of the high commercially valued farm fishes which have expanded tremendously in the northern part of Bangladesh. However, despite its impressive growth in the initial years, output of the industry is highly volatile across different years and farms. This research, following the Just and Pope framework, has made use of Kumbhakar’s (2002 Kumbhakar, S.C. (2002) Specification and estimation of production risk, risk preferences, and technical efficiency. American Journal of Agricultural Economics, 84, 8–22.[Crossref], [Web of Science ®] , [Google Scholar]) extended stochastic frontier model (SFM) to estimate production risk and technical efficiency (TE) in Thai koi farming. Results show that feed is the most important input significantly influencing production. Zeolite and pesticide appear to be risk-reducing, while fingerlings, labor, feed and salt turn out to be risk-increasing inputs. The inefficiency model indicates that labor and pond area significantly reduce the technical inefficiency. Feed, fingerling and lime also act as TE-improving factors. Education of farmers improves TE, while experience, training and frequency of water change do not impact the TE. The mean TE scores are 0.73 and 0.96 for the flexible risk estimate and conventional SFM estimates, respectively. TE values appear to become exaggerated when production risk is excluded from the model. SFM recognizes that risk is involved in Thai koi farming that causes lower TE.     

锦鲤的疾病防治

锦鲤属鲤形目、鲤科、鲤亚科、鲤属,现已有100多个品种。由于锦鲤体格健壮,游姿高雅,体型优美,色彩艳丽,斑纹灿烂,适应能力强,目前在我国不论是公园的水塘,还是家庭的鱼缸,到处都可以见到锦鲤矫健而美丽的身影。在养殖过程中,锦鲤的疾病和大多数鱼类一样,由于发现疾病症状较晚而难以治     

锦鲤疱疹病毒组织灭活疫苗研制初探

本研究的目的是制备针对锦鲤疱疹病毒的组织浆灭活疫苗,用来控制目前集约化养殖鲤鱼大面积暴发的锦鲤疱疹病毒。利用现场采集的病死鱼内脏,匀浆后腹腔注射进行攻毒试验,采集攻毒后病死鱼的内脏匀浆后,加入0.5%甲醛制备组织浆灭活疫苗。确定所制疫苗无杂菌污染后,在实验室内对6组实验鱼采用不同的接种方法进行疫苗接种,同时设立阴性对照组。实验结果表明,所得疫苗可以在一定程度上降低死亡率,但不能100%的控制发病,具体原因有待进一步探讨。     

Morphogenesis of koi herpesvirus observed by electron microscopy

Koi herpesvirus (KHV or cyprinid herpesvirus 3) was inoculated onto three fish cell lines derived from carp, Cyprinus carpio L., and the process of virion formation observed with electron microscopy. Essentially, similar features of virus particles were observed in all three cell lines. The nucleus of infected cells was characterized by margination of chromatin and often contained many capsids of about 110 nm in diameter with varying morphology. The morphological variation of the capsids was very similar to that of mammalian herpesviruses. Some capsids protruded from the inner nuclear membrane, and others, with envelopes, were located in the perinuclear space between the inner and outer nuclear membrane, suggesting budding of capsids at the inner nuclear membrane. Naked capsids and envelopment of capsids by budding into vesicles were also observed in the cytoplasm. Mature, enveloped virions 170-200 nm in diameter were seen in cytoplasmic vesicles or outside the cell. These observations suggest KHV virions mature through a complex morphological pathway including two distinct envelopments, which have been found only in herpesviruses. These observations support the inclusion of KHV in the family Herpesviridae.     

热应激对大正三色锦鲤非特异性免疫指标及HSP70基因表达的影响

为探讨锦鲤的抗逆机理,在实验室条件下研究了持续热应激对大正三色锦鲤非特异性免疫指标及HSP70基因相对表达量的影响.分别于应激前、应激后2、6、10、14、18、22、26 h进行取样测定呼吸爆发、补体蛋白3(C3)、超氧化物歧化酶(SOD)、丙二醛(MDA)以及热休克蛋白70 (HSP70)基因的相对表达量.结果发现,热应激后呼吸爆发降低,在应激后2、10、18～26 h降低显著(P＜0.05);热应激2h后血清中C3含量略有上升,应激6～26 h过程中与应激前相比均下降,应激14 ～ 22 h显著地下降(P＜0.05);热应激下血清中SOD有上升的趋势,但差异不显著(P＞0.05);热应激下,血清中MDA浓度升高,应激10～18 h上升显著(P＜0.05);应激后2、26 hHSP70基因的相对表达量与应激前相比显著性地上升了5.93倍、2倍(P＜0.05),应激后6～22 hHSP70基因的相对表达量与应激前水平无显著性差异(P＞0.05).结果表明,热应激影响锦鲤非特性免疫指标,降低锦鲤非特异性免疫力;HSP70的表达受热应激调节,热应激下诱导合成的HSP70对锦鲤起到一定的应激保护作用.     

Endocrine disrupting effects of 4-nonylphenol on plasma vitellogenin,reproductive system and histology in koi carp (Cyprinus carpio)

International Aquatic Research - This study aimed to assess the effect of 4-nonylphenol (4-NP), a ubiquitous aquatic micro-chemical pollutant substance on some on some reproductive parameters and...