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本研究建立了定量检测鲤疱疹病毒2型(Cyprinid herpesvirus 2,CyHV-2)的微滴式数字PCR(Droplet digital PCR,ddPCR)检测方法,并与实时荧光定量PCR(Quantitative real-time PCR,qPCR)检测方法的灵敏性、重复性、特异性和临床样品检测做了比较分析.结果表明,与qPCR相比,ddPCR具有相同的特异性,其灵敏性比qPCR低20倍.在定量CyHV-2 DNA时,ddPCR (R2=0.994)和qPCR (R2=0.994)均表现出良好的线性关系,且2种检测方法间的定量值呈正相关(R2=0.989).在定量检测相同稀释度的CyHV-2 DNA时,qPCR的定量值始终比ddPCR高10倍.ddPCR的组内和组间重复变异系数(CV)分别为0.59％-11.26％和6.55％-23.21％,而qPCR为16.57％-27.56％和22.31％-56.73％,说明ddPCR具有更好的稳定性.在临床样品定量检测时,ddPCR的检出率稍高于qPCR.本研究建立的ddPCR能够准确定量检测CyHV-2,将为CyHV-2相关研究提供有益参考. 相似文献
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The distribution and expression of lymphocystis disease virus (LCDV) vaccine, on the basis of DNA vaccine (pEGFP-N2-LCDV0.6 kb) construction, were analyzed in tissues of the Japanese flounder by PCR, RT-PCR and fluorescent microscopy. Results from PCR studies indicated that the vaccine-containing plasmids were distributed in injected muscle, muscle located opposite the injection site, hind intestine, gill, spleen, head kidney, liver and gonad 7 days after vaccination. However, these vaccine-containing plasmids disappeared by 90 days following vaccination. Fluorescent microscopy observations revealed that green fluorescence appeared in muscle, muscle located at the opposite side of the injection site, hind intestine, gill, spleen, head kidney and liver of fish 36 h after vaccination, and that green fluorescence did not appear in control tissue. The green fluorescence became weaker at 60 days post-vaccination, however, it remained detectable in the spleen 90 days post-vaccination. Results from RT-PCR studies indicated that the Mcp gene is expressed in all tissues of vaccinated fish 7-20 days after vaccination. These results demonstrate that the DNA vaccine is distributed and expressed in different tissues of vaccinated fish, and therefore, may have provided an antigen producing specific immune response. 相似文献
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Herpesviral haematopoietic necrosis is a disease of goldfish, Carassius auratus , caused by Cyprinid herpesvirus-2 (CyHV-2) infection. Quantitative PCR was carried out on tissue homogenates from healthy goldfish fingerlings, broodfish, eggs and fry directly sampled from commercial farms, from moribund fish submitted to our laboratory for disease diagnosis, and on naturally-infected CyHV-2 carriers subjected to experimental stress treatments. Healthy fish from 14 of 18 farms were positive with copy numbers ranging from tens to 107 copies μg−1 DNA extracted from infected fish. Of 118 pools of broodfish tested, 42 were positive. The CyHV-2 was detected in one lot of fry produced from disinfected eggs. Testing of moribund goldfish, in which we could not detect any other pathogens, produced 12 of 30 cases with 106 –108 copies of CyHV-2 μg−1 DNA extracted. Subjecting healthy CyHV-2 carriers to cold shock (22–10 °C) but not heat, ammonia or high pH, increased viral copy numbers from mean copy number (±SE) of 7.3 ± 11 to 394 ± 55 μg−1 DNA extracted after 24 h. CyHV-2 is widespread on commercial goldfish farms and outbreaks apparently occur when healthy carriers are subjected to a sharp temperature drop followed by holding at the permissive temperature for the disease. 相似文献
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急性病毒性鲫鳃出血病的病理变化 总被引：3，自引：0，他引：3
病毒性鲫(Carassius auratus)鳃出血病的突发流行可导致鱼高致死率,使中国部分养鱼场近些年受到重创。为了充分认识这种鱼病的发生发展过程,我们结合PCR、光学显微镜和透射电镜,对病鱼头肾等组织的病变及病毒分布情况进行了分析。注射自然发病鱼的组织滤液(病毒悬液),能使正常鲫感染,且出现与自然发病鲫相同的症状及高致死率;病原有典型疱疹病毒的形态特征(故称为鲫疱疹病毒,Ca HV)。经PCR扩增对已知鲤科疱疹病毒主要衣壳蛋白(MCP)基因进行检测,可确定Ca HV存在于被感染鱼的肝、脾、肾和头肾组织中。对组织病理变化及不同时序进行比较,结果表明Ca HV感染会引起鲫这些组织不同程度病变,而鳃和头肾的病理变化有显著时序相关性。在头肾细胞中还观察到大量Ca HV颗粒,预示鲫头肾是Ca HV侵染和复制的主要靶器官。 相似文献
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混养的异育银鲫和鲢鱼种大批死亡,为明确发病死亡的病原和组织损伤并提供相关的疾病防控措施,进行了病鱼肉眼和显微镜检查、细菌学检测、病毒学检测、组织病理和药敏试验研究。结果发现,除在患病鱼体表偶然发现有少量不会引起充血等症状的杯体虫和车轮虫外,未在体内外发现其他寄生虫和真菌类病原;通过细菌分离、人工回感试验、生理生化特性和16S r RNA基因序列分析,从患病异育银鲫和鲢分离到的致病菌株均为嗜水气单胞菌;根据异育银鲫和鲢病毒性疾病的现状,使用鲤疱疹病毒2型(Cyprinid herpesvirus 2,Cy HV-2)DNA聚合酶基因的特异性引物分别对自然发病的异育银鲫和鲢进行PCR检测,只有异育银鲫检测到Cy HV-2,分别用它们的除菌组织上清液进行人工感染试验,只有异育银鲫出现充血症状和死亡现象;由此得出嗜水气单胞菌是异育银鲫和鲢发病死亡的主要病原,Cy HV-2是异育银鲫混合感染的次要病原。患病鲢与患病异育银鲫呈现出类似的组织病理现象,又有一些各自特有的组织病理表现,单纯细菌感染的鲢轻度病变以细胞颗粒变性为主,坏死细胞以核溶解为主,细菌和病毒混合感染的异育银鲫肝脏轻度病变以细胞滴状玻璃样变的变性为主,坏死组织细胞以核固缩和核碎裂为主,在肾脏和脾脏出现染色质边集于核膜的肿大细胞核,主要组织器官出现从变性到坏死的病理变化过程,最终失去应有的功能而死亡。依据药敏试验结果,建议内服诺氟沙星和氟苯尼考等抗生素防治本病的嗜水气单胞菌感染,混合感染Cy HV-2的异育银鲫可以通过注射Cy HV-2疫苗和生态养殖的方法控制和减少该病毒病感染和发展。 相似文献
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对我国虹彩病毒感染的大菱鲆Scophthalmus maximus进行的组织病理和超微病理学研究发现,该病典型的病理学特点是在病鱼的脾脏、肾脏、肠、肝脏、鳃、心脏和皮肤等器官组织内出现嗜碱性的肿大细胞。病毒感染导致患病大菱鲆多个器官组织发生了不同程度的病理变化,其中以脾脏组织的病理变化最为显著,表现为造血组织的严重坏死。此外,肾脏造血组织发生坏死、肠固有膜和黏膜下层出血和水肿、肝细胞水样变性、心肌局灶性坏死以及皮肤真皮层出血并伴有水肿和炎性渗出也是该病常见的组织病理学变化。超微病理研究表明,肿大细胞内有虹彩病毒粒子存在。病毒分布于受感染细胞的胞质、组织间隙以及血管腔内。受感染细胞出现线粒体和内质网等细胞器肿胀、崩解等细胞病理变化。研究认为,病毒感染造成皮下组织血管损伤出血,是虹彩病毒感染的大菱鲆发生＂红体病＂的原因所在。虹彩病毒感染所致的机体严重贫血是患病大菱鲆死亡的主要原因,而主要器官组织的病变使得病鱼器官功能衰竭则可加速鱼的死亡。 相似文献
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Lymphocystis disease is a prevalent, non-fatal disease that affects many teleost fish and is caused by the DNA virus lymphocystis disease virus (LCDV). Lymphocystis-like lesions have been observed in yellow perch, Perca flavescens (Mitchell), in lakes in northern Alberta, Canada. In an effort to confirm the identity of the virus causing these lesions, DNA was extracted from these lesions and PCR with genotype generic LCDV primers specific to the major capsid protein (MCP) gene was performed. A 1357-base pair nucleotide sequence corresponding to a peptide length of 452 amino acids of the MCP gene was sequenced, confirming the lesions as being lymphocystis disease lesions. Phylogenetic analysis of the generated amino acid sequence revealed the perch LCDV isolate to be a distinct and novel genotype. From the obtained sequence, a real-time PCR identification method was developed using fluorgenic LUX primers. The identification method was used to detect the presence/absence of LCDV in yellow perch from two lakes, one where lymphocystis disease was observed to occur and the other where the disease had not been observed. All samples of fin, spleen and liver tested negative for LCDV in the lake where lymphocystis disease had not been observed. The second lake had a 2.6% incidence of LCD, and virus was detected in tissue samples from all individuals tested regardless of whether they were expressing the disease or not. However, estimated viral copy number in spleen and liver of symptomatic perch was four orders of magnitude higher than that in asymptomatic perch. 相似文献
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Steinum T Kvellestad A Rønneberg LB Nilsen H Asheim A Fjell K Nygård SM Olsen AB Dale OB 《Journal of fish diseases》2008,31(3):205-214
11.
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。 相似文献
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Smail DA Bain N Bruno DW King JA Thompson F Pendrey DJ Morrice S Cunningham CO 《Journal of fish diseases》2006,29(1):31-41
During mid-June 1999 peak mortalities of 11% of the total stock per week were seen at a sea cage site of Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland. Virus was isolated on chinook salmon embryo (CHSE) cells in a standard diagnostic test and infectious pancreatic necrosis virus (IPNV) identified by enzyme-linked immunosorbent assay. IPNV was confirmed as serogroup A by a cell immunofluorescent antibody test using the cross-reactive monoclonal antibody AS-1. Four weeks after the main outbreak, virus titres in surviving moribund fish were assayed at >10(10) TCID50 g(-1) kidney. Histopathology of moribund fish was characterized by pancreatic acinar cell necrosis and a marked catarrhal enteritis of the intestinal mucosa. In the liver, necrosis, leucocytic infiltration and a generalized cell vacuolation were noted. IPNV-specific immunostaining was demonstrated in pancreas, liver, heart, gill and kidney tissue. The nucleotide sequence of the coding region of segment A was determined from the Shetland isolate. A 1180 bp fragment of the VP2 gene of this isolate was compared with a 1979 reference isolate from mainland Scottish Atlantic salmon, La/79 and another more recent mainland isolate, 432/00. Both A2 isolates were derived from carrier fish without signs of IPN and serotyped by a plaque neutralization test. The Shetland isolate shows a different nucleotide and amino acid sequence compared with the two isolates from carrier fish. These latter isolates showed identical amino acid sequences in the fragment examined, despite the 21 years separating the isolations. Sequence comparisons with other A2 (Sp) isolates on the database confirm all three Scottish isolates are A2 (Sp). 相似文献
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Abstract. In a study of the possible role of waterborne infectious haematopoietic necrosis virus in transmission of the disease among spawning sockeye salmon, Oncorhynchus nerka (Walbaum), both infection rates and virus titres were higher in fish held at high density in a side channel than in fish in the adjacent river. Virus was never isolated from river water, but was found in water from the side channel at levels ranging from 32.5 to 1600 plaque-forming units (p.f.u.)/ml. Uninfected yearling sockeye salmon held in a box in the side channel developed localized gill infections with IHN virus. The disease did not progress to the viscera until a threshold titre of about 105 p.f.u./g was reached in the gill. The effectiveness of the gill as a barrier limiting development of systemic infections means that waterborne IHN virus probably does not greatly increase the infection rate in a sockeye salmon population during spawning. 相似文献
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Atlantic cod, Gadus morhua , averaging 100 g, were experimentally challenged by intraperitoneal injection of nervous necrosis virus (NNV) originating from Atlantic halibut. Cod tissues, including blood, gill, pectoral fin, barbel, ventricle, atrium, spleen, liver, lateral line (including muscle tissue), eye (retina) and brain, were sampled at day 25 and 130 and investigated by real-time RT-PCR for the presence of NNV. Relative quantifications at day 130 were calculated using the 2−ΔΔCt method. Immunosuppression by injection of prednisolone-acetate was introduced for a 30-day period, and tissue sampled at day 180 and relative quantification estimated. No mortality or clinical signs of disease were observed in the challenged group. The challenge resulted in detection of NNV in blood, spleen, kidney, liver, heart atrium and heart ventricle at day 25, and by the end of the experiment NNV showed a clear increase in brain and retina, suggesting these to be the primary tissues for viral replication. There was no increase in the relative amount of NNV in blood, atrium, ventricle, spleen, liver and kidney. Corticosteroid implants resulted in a weak increase in virus RNA in spleen, kidney, liver and brain. These findings suggest that Atlantic cod is susceptible to infection with NNV from halibut. The observed tissue tropism patterns suggest an initial viraemic phase, followed by neurotrophy. Head-kidney is the best tissue identified for possible NNV detection by non-lethal biopsy, but detection was not possible in all injected fish. 相似文献
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A synthetic antibacterial peptide from Mytilus galloprovincialis reduces mortality due to white spot syndrome virus in palaemonid shrimp 总被引：3，自引：0，他引：3
White spot syndrome virus (WSSV) isolated from Penaeus monodon was found to be highly infective for the western Mediterranean shrimp, Palaemon sp. Using polymerase chain reaction (PCR), it was demonstrated that such shrimp are not naturally carriers of WSSV. Following challenge with virus, mortality reached 100% 3.5-4 days after injection at 22 degrees C. Incubation of infected shrimp at 10 degrees C totally suppressed the mortality which rapidly developed when shrimp were returned to 18 or 22 degrees C. Preincubation of WSSV with mature synthetic mytilin significantly reduced shrimp mortality with a 50% efficient dose of about 5 microM. Survival of shrimp was not due to the development of an active mechanism of defence as re-injection of WSSV produced the same mortality pattern. Mortality was probably due to WSSV replication as dot blot failed to detect viral DNA in the injection sample but was positive 1 day post-injection. Protection by mytilin was by interaction at the virus level, preventing replication as no WSSV nucleic acid was detected by PCR even after 7 days in shrimp injected with WSSV preincubated with 10 or 50 microM mytilin. 相似文献
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Two new cell cultures from flounder, Paralichthys olivaceus (Temminck & Schlegel), flounder fin (FFN) cells from fin tissue and flounder spleen (FSP) cells from spleen tissue, were established and characterized. The cells multiplied well in Eagle's minimum essential medium, supplemented with 10% foetal bovine serum, and have been subcultured more than 100 times, becoming continuous cell lines. Modal diploid chromosome number of FFN and FSP cells was 64 and 62, respectively. Polymerase chain reaction products were obtained from FFN and FSP cells with primer sets ofmicrosatellite markers of flounder. Optimal growth temperature was 20 degrees C and consisted of epithelioid cells. FFN and FSP cells showed cytopathic effects after inoculation of infectious pancreatic necrosis virus, marine birnavirus, chum salmon virus, infectious haematopoietic necrosis virus, spring viraemia of carp virus and hirame rhabdovirus. Thus these new cell lines may be useful for studying a wide range of fish viruses. 相似文献
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This study investigates the occurrence and distribution pattern of infectious pancreatic necrosis virus (IPNV) within the pancreas, liver, kidney and spleen of naturally infected cultured rainbow trout, Oncorhynchus mykiss (Walbaum), using immunohistochemistry (IHC). A nested PCR was also employed to confirm the presence of the virus in the pooled tissues of the specimens. All the examined tissues except spleen were immunohistochemically positive for IPNV, but staining intensity and distribution pattern varied. The kidney tubules had the most intense and widespread staining by IHC, indicating a specific tissue tropism at least for this particular serotype. The nucleotide sequence had the greatest identity with the Sp serotype confirming the presence of the nucleic acid of IPNV in the pooled tissues. Based on the present findings, it could be concluded that the absence of lesions consistent with infectious pancreatic necrosis (IPN) disease in the H&E‐stained sections cannot rule out the presence of the IPNV, and the use of an alternative rapid confirmatory method such as IHC with formalin‐fixed, paraffin‐embedded tissue sections is helpful for the final diagnosis of IPN in rainbow trout. 相似文献
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2016年4月,四川某鲈鲤(Percocypris pingi)养殖场流行一种以鳃、鱼鳔和内脏器官出血为临床特征的传染病。组织病理学观察发现,患病鲈鲤全身多组织器官均发生明显的病理损伤,尤其是肝、脾、肾、鳃和肠表现为明显的出血、变性、坏死以及炎症细胞浸润。取病鱼组织匀浆滤液接种鲤上皮瘤细胞(epithelioma papulosum cyprini, EPC),盲传3代出现典型的细胞病变(cytopathic effects, CPE)。将自然发病鱼组织匀浆滤液和细胞培养病毒液分别接种健康鲈鲤,实验鱼出现与自然发病鱼相同的症状,死亡率分别为60%和50%,而对照组未见异常。对经分离毒株ZLP160415感染出现CPE的EPC细胞制备超薄切片进行电镜观察,发现病毒呈弹状,长90～150 nm,宽40～60 nm;对自然发病鱼、人工感染发病鱼内脏组织和细胞培养病毒液进行鲤春病毒血症病毒(springviremiaofcarpvirus,SVCV)的RT-PCR检测,均扩增出目的条带。基于SVCV糖蛋白基因进行系统发育分析,结果显示分离毒株(ZLP160415)属于Ia型。结合本次疾病的流行病学与病理损伤特点、病毒分离鉴定、人工感染试验结果和透射电镜检查,确定此次流行病的病原为SVCV。 相似文献
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一种基于Ⅱ型鲤疱疹病毒衣壳蛋白72的免疫学检测方法 总被引：3，自引：1，他引：3
Ⅱ型鲤疱疹病毒(cyprinid herpesvirus 2,Cy HV-2)是引起养殖异育银鲫(Carassius auratus gibelio)造血器官坏死症的致病病原。在临床筛查中基于病毒核酸的PCR和real time PCR技术已经建立,但是稳定性更强的免疫学诊断技术国内外尚无报道。本研究目的是利用Cy HV-2编码的ORF72基因(Gen Bank登录号:AFJ20502.1)所编码的衣壳蛋白作为捕获抗原,通过识别感染病毒的鱼体中的相应抗体,从而对样本进行临床免疫学检测。首先采用PCR方法从纯化的Cy HV-2基因组中扩增ORF72基因,并把该基因克隆至原核表达载体PGEX-4T-3,并转化到大肠杆菌中诱导表达,诱导表达的产物通过SDS-PAGE进行鉴定,对表达的重组蛋白进行纯化。用已纯化的72重组蛋白对小鼠进行免疫,制得72重组蛋白的抗体。Western blot检测表明所制备的多克隆抗体既能识别原核表达的重组蛋白,也可以识别Cy HV-2病毒粒子上的衣壳蛋白72。在上述基础上建立了基于Western blot技术的Cy HV-2抗体检测技术:用纯化的72重组蛋白作为检测抗原,鲫鱼血清用作一抗,兔抗鲫Ig M多克隆抗体作为二抗,酶标羊抗兔作为三抗鉴定鲫鱼是否存在Cy HV-2特异性抗体。在对急性感染期的临床样本检测中,本方法能在所有样本中检测出ORF72特异性抗体存在,表明72重组蛋白作为相应抗体捕获原可以用于确诊鲫鱼是否感染Cy HV-2。本研究建立的实验室免疫学检测方法为商品化免疫学检测技术的开发奠定了基础,对Cy HV-2的检验检疫具有一定的临床应用价值。 相似文献
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为了查明鲤疱疹病毒Ⅱ型(Cyprinid herpesvirus 2,Cy HV-2)的理化与生物学特性以及病毒在细胞内的超微形态发生过程,利用新建立的对Cy HV-2敏感的异育银鲫脑组织细胞系(Gi CB),对Cy HV-2的理化及生物学特性进行了详细研究,比较了不同来源鱼类细胞系对Cy HV-2感染的敏感性,并对体外培养细胞中Cy HV-2病毒粒子及其超微形态发生过程进行了电镜观察。结果显示,Cy HV-2对热、酸、碱、有机溶剂和冻融敏感;常用鱼类细胞系EPC、RTG-2、Koi-Fin、CIK、CCK、PF-Fin对Cy HV-2的感染不敏感,特异性巢式PCR检测盲传至第7代Cy HV-2细胞培养物,结果均为阴性;Cy HV-2在Gi CB细胞中的增殖动态研究结果表明:病毒感染细胞经过12 h的隐晦期,24 h开始进入对数生长期,96 h病毒滴度达到最高值(107.52±0.26 TCID50/m L),然后进入平台期;透射电子显微镜观察结果显示,Cy HV-2感染细胞可分为吸附与侵入、复制与装配、成熟与释放3个主要过程,病毒进入对数生长期后,被感染细胞内可见形态典型的疱疹病毒颗粒。 相似文献