Superinfection in ducks persistently infected with duck plague virus

Superinfections with homologous or heterologous strains of duck plague virus resulted in the deaths of birds persistently infected with duck plague virus. Not all birds that were superinfected died. Protection against mortality depended on the route of exposure, strain of the initial duck plague virus, and strain of the superinfecting virus.     

鸭瘟病毒强毒株感染SPF鸭后动态病理组织学变化

用鸭瘟病毒(Duck plague virus,DPV)人工感染2月龄SPF鸭,定期剖杀,经聚合酶链反应(PCR)检测为鸭瘟后,对各组织器官的病理组织学变化进行观察,并进行血常规和血液生化指标检测.结果显示,人工感染后24 h,试验鸭中枢免疫器官胸腺、法氏囊表现为淋巴细胞数量减少,组织间隙增大;肝脏、脾脏组织病变较为严重,大部分组织器官均出现程度较轻的病理变化.感染后48～96 h,中枢免疫器官的淋巴细胞极度减少、网状细胞增生、组织器官结构模糊不清,严重充血、出血;其余组织器官出现细胞变性、出血等不可逆病理变化.感染后120 h,组织细胞变性、坏死,出现大片坏死区.点眼滴鼻组鸭感染DPV后组织学变化与皮下注射组相似,只是发生的时间偏后约24～48 h.对照组鸭病理组织学观察未见损伤.WBC、HGB、AST、ALT等发生显著变化.结果表明,接种DPV强毒感染鸭的组织器官严重受损,特别是免疫器官,甚至会引起免疫抑制.     

不同条件对鸭瘟病毒感染细胞蛋白质组二维电泳分析结果的影响

本研究以鸭瘟病毒感染鸭胚成纤维细胞为材料,围绕影响二维电泳因素进行全面探讨,以建立和优化鸭瘟病毒感染细胞蛋白质组二维电泳模型.结果表明,样品经过冷丙酮处理,水化液DTT浓度为30 mmol/L都有利于等电聚焦;采用PH5～8 IPG窄胶条和混合两性裁体电解质pH3～10/pH5～8为2/1比pH3～10 IPG宽胶条和单一两性载体电解质PH3～10分离蛋白时,各蛋白点间距较大,分辨率高,更有利于显示低丰度蛋白点;1.5 mg的蛋白上样量偏大,2～DE图像出现拖尾和水平条纹,部分相邻高丰度的蛋白重叠,且还掩盖了低丰度蛋白点.PDQuest7.40软件分析显示:17 cm PH5～8 IPG胶条电泳鸭瘟病毒感染细胞蛋白质组,银染可获得1 253个蛋白点,而考染却检测到388个蛋白点;重复试验仍获得清晰、稳定的2-DE图像,同一样本不同时期,考染可获得约348、331个蛋白点,蛋白点匹配率达88%,表明了鸭瘟病毒感染细胞蛋白质组二维电泳模型稳定、分辨率高、重复性好,为鸭瘟病毒蛋白组的进一步研究和新蛋白的发现提供了重要的研究方法.     

鸭瘟病毒在雏鸭体内的动态定量分布及其潜伏部位研究

为研究鸭瘟病毒的组织细胞嗜性及其潜伏部位,采用real-timePCR技术对人工感染后病毒的组织器官分布进行了动态定量分析。结果表明,鸭瘟病毒在肝、脾、外周血淋巴细胞、法氏囊、三叉神经节、肾、肺和心脏等均能增殖;动态定量分析发现,随疾病发展各器官病毒荷载量不断上升,至死亡时达到顶峰;肝、脾中病毒载量高,出现时间早,持续时间长;耐过鸭多数组织器官中病毒逐渐消失,但三叉神经节及外周血淋巴细胞在感染后38d仍能检测到低拷贝病毒DNA,表明除三叉神经节外,外周血淋巴细胞也很可能是潜伏部位。     

A sequential histologic and immunohistochemical study of duck hepatitis B virus infection in Pekin ducks

Twenty-nine Pekin ducks were inoculated with duck hepatitis B virus (DHBV), DHBV-free serum, or saline at 1 day of age. Congenitally DHBV-infected ducks were also studied. Ducks were killed periodically during a 92-week study and examined histologically and immunohistochemically to assess liver and extrahepatic inflammation and to detect and characterize DHBV core antigen tissue distribution. DHBV infection produced an asymptomatic but persistent DHBV viremia in all ducks associated with a mild to moderate transient hepatic inflammation apparent at 3 to 6 weeks post-inoculation and waning afterwards. DHBV core antigen was detected in hepatocyte cytoplasm at 1 week post-inoculation, and by 3 weeks post-inoculation scattered pancreatic acinar and islet cells also contained viral antigen. Small numbers of mononuclear cells in the splenic white pulp also contained viral antigen. Viral antigen persisted in all of these tissues throughout the duration of the experiment. No inflammation or tissue injury was detected in any of the extrahepatic tissues during the course of DHBV infection. One DHBV-injected duck developed a hepatocellular carcinoma at 88 weeks of age. Isolated patches of neoplastic hepatocytes contained cytoplasmic DHBV core antigen. The results of this study indicate that DHBV, like mammalian hepadnavirus, is capable of producing a persistent infection of the liver and several extrahepatic tissues and suggest that persistent infection may be associated with the development of hepatocellular carcinoma.     

Pathological and immunological study of goose embryos experimentally infected with duck plague virus

A total of 240 embryonated goose eggs obtained from two susceptible flocks were used. Half of the eggs were inoculated into the allantoic cavity with a virulent strain (7593) of duck plague virus isolated from an acute outbreak, and the other half were inoculated with the attenuated vaccine virus (KAPEVAC). Ten, 100 or 1000 CPU/0.1 ml virus were given on days 12 and 20 of incubation. Embryos that died and surviving embryos killed at 5-day intervals were examined by light and electron microscopy. The yolk and the serum of embryos that survived until hatching were assayed for antibody content. Lymphocytes separated from the blood were used for the immuno-rosette formation and lymphocyte stimulation tests. Pathomorphological changes indicative of virus replication occurred in the liver, kidney, myocardium, gizzard muscle and chorioallantoic membrane (CAM) of the embryos in the case of both virus strains. The time of onset and severity of these changes and the time and rate of embryonic mortality depended on the virulence of the strain used for inoculation, the virus dose and the time of inoculation. Virus-neutralizing (VN) antibodies were demonstrable neither in the yolk nor in the serum of goslings exsanguinated after hatching. The lymphocytes recognized the virus antigen in the in vitro cellular tests and responded to it with blastogenic transformation. As opposed to adult birds, in the embryos duck plague virus infection did not cause damage to the digestive tract mucosa and the lymphoid organs.     

商品肉鸭鸭瘟病毒的分离与鉴定

采用鸭胚成纤维细胞培养从山东和北京两地暴发的鸭瘟临床病例中分离到两株鸭肠炎病毒（DEV），分别命名为SD和BJ。以单抗介导的间接免疫荧光（IFA）检测方法，对两个分离株感染细胞滴片进行间接IFA检测，可见感染细胞内有明显的蓝绿色荧光。试验感染7日龄北京鸭可引起鸭瘟的典型临床症状．死亡率为100％（3／3），取试验感染死亡鸭肝脏、法氏囊和脑组织等制备石蜡包埋切片，利用单抗进行免疫组化检验，除脑组织外均检测到病毒抗原。根据在GenBank上已发表的DEV两段序列设计两对引物，采用聚合酶链式反应（PCR）对野毒sD株人工感染鸭肝脏和BJ珠自然发病鸭肝脏病科提取核酸为模板进行扩增，得到预期大小为765bp和1954bp的目的片段，对长片段进行测序，与发表序列进行比较，毒株间的碱基序列同源性达到99．73％。     

Isolation from ducks of a hypervirulent strain of duck plague virus and an avian type 6 paramyxovirus

This paper describes the isolation and identification of a duck plague virus (DP) and a paramyxovirus (PMV₆), from the livers and intestines collected in 4-month old mule ducks, under fattening, exhibiting 75% mortality and necrotic-haemorrhagic gross lesions. These viruses were isolated in specific pathogen free (SPF) muscovy duck eggs and SPF chicken eggs respectively. Then the DP virus was adapted to duck and chicken fibroblasts. The disease was reproduced in 2-week old SPF muscovy ducklings, intramuscularly inoculated with the previous organs, as well as in contact ducks. From them, only the DP virus was isolated again. Experimentally the intramuscular inoculation of the duck plague French vaccinal strain, 4 h post contact, did not prevent the disease and did not decrease its severity.

Regarding the DP virus, the typical signs and lesions observed in experimentally infected muscovy ducks as well as the presence of intranuclear inclusions of the epithelial cells of their oesophagus, intestines, bursa of Fabricus and liver on the one hand, and on the other hand, of the epithelial cells of the duck egg chorio-allantoïc membrane and fibroblasts inoculated with the samples first defined, allowed the characterization of the virus. Direct electron microscopy, as well as the results of seroneutralization tests with different specific avian Herpes virus antisera confirmed the DP virus identification. Moreover the DP isolate was not antigenically different from the serotype actually known.

The haemagglutinating virus (PMV₆) was characterized by direct electron microscopy as well as with 18 specific avian Myxovirus antisera; its identification was confirmed too by the specific seroconversion observed 4 weeks post-inoculation of this virus, in 11 weeks old SPF muscovy ducklings.

Finally an assay was carried out to appreciate the pathogenicity of theses viruses inoculated either separately or associated. It showed the high pathogenicity of the DP strain. The PMV₆ was apathogenic and no synergic effect with the DP virus was demonstrated. It appears to be the first isolation of PMV₆ in France, to our knowledge. The epidemiological circumstances related to theses isolations are discussed. The failure of the emergency vaccination in contact ducks, might be attributed to the high virulence of the DP strain.     

应用TaqMan-MGB探针FQ-PCR探讨鸭瘟弱毒苗在鸭体内的分布及排泄规律

用鸭瘟病毒(DPV)Cha株弱毒苗皮下注射免疫20日龄樱桃谷鸭,应用TaqMan-MGB探针实时荧光定量PCR(FQ-PCR)对免疫后10、30、609、0 min及12、24 h和6、9、12、21、366、0 d鸭的心、肝、脾、肺、肾、脑、胸腺、法氏囊、哈德氏腺、回盲处、血液、气管、气管分泌液、食管及其分泌液、十二指肠、空肠、回肠、肓肠、直肠及各肠段分泌液中DPV-DNA的拷贝数(以对数值表示)进行了检测。结果显示,免疫后10 min即可在胸腺和血液中检测到DPV-DNA;60 min肾脏中DPV-DNA拷贝数最高(9.349);脾(9.932)、脑(9.791)、胸腺(9.736)、法氏囊(9.598)及哈德氏腺(8.135)中DPV-DNA拷贝数于90 min分别达到最高;12 h后所有受检样品中都能检测到DPV-DNA,其中心脏中DPV-DNA拷贝数最高(9.818);肠道中DPV-DNA拷贝数在免疫后6~12 d明显高于其他组织,其中盲肠是DPV-DNA拷贝数(10.591)最高的受检样品,直肠分泌液中DPV-DNA最早于90 min检测到。研究表明,免疫鸭于免疫早期在包括主要免疫器官在内的各实质...     

鸭瘟病毒强毒株在感染鸭实质器官内的增殖与分布

鸭瘟病毒(DPV)CHv强毒株经皮下注射、滴鼻和口服3种途径分别感染20日龄天府肉鸭,于攻毒后10、30、60、90min以及4、12、48、72h和9、15d每组分别剖杀2只鸭,采集心、肝、脾、肺、肾、脑、胸腺、法氏囊、哈德氏腺等实质器官,应用TaqMan-MGB探针实时荧光定量PCR对DPV在这些器官的分布和增殖进行检测。结果表明,DPV分布到具体器官的速度与感染的途径、鸭的解剖结构密切相关,其中皮下注射是DPV分布到各实质器官速度最快的途径。30min于皮下感染鸭的肝、脾、胸腺、法氏囊、哈德氏腺、肺、脑、肾,口服感染鸭的肺和法氏囊,滴鼻感染鸭的心脏和哈德氏腺均检测到DPV-DNA;90min所有受检样品中检测到DPV-DNA。鸭抗DPV感染的免疫器官的重要性依次是脾、胸腺、法氏囊和哈德氏腺,30min内DPV-DNA分布到脾、胸腺、法氏囊的速度和数量决定了DPV感染的潜伏期和疾病的严重程度。不同途经感染鸭的相同器官在同一时间内的DPV-DNA拷贝数大多以皮下感染鸭为最高。DPV致死鸭的法氏囊和肾是DPV-DNA含量最高的实质器官。     

鸭瘟病毒的分离和鉴定

1999年 8月广西玉林的养鸭大户送来一批一周龄左右的死亡雏鸭 ,称其养的 2 0 0 0多只雏鸭死亡过半 ,药物治疗不能控制病情。经临床剖检病变很象小鸭肝炎 ,我们采集了几份死鸭肝病料进行分离鉴定 ,现将结果报告如下。1　试验材料和方法1 .1　材料鸭瘟病毒阳性血清、Ⅰ型鸭肝炎病毒阳性血清由中国农业大学苏敬良博士惠赠。鸭胚与2日龄雏鸭购自本市健康鸭场孵化场。1 .2　病料处理对病鸭的肝脏先进行细菌分离 ,镜检。然后按常规方法作成 1 :5乳剂 ,离心取上清液 ,检验无菌后 ,-2 0℃保存备用。1 .3　病毒分离将处理好的病料经尿囊腔接种 1 1日…     

鸭副粘病毒人工感染鸭的病理组织学研究

为研究鸭副粘病毒致病机理及病理组织变化。本研究将20日龄非免疫健康建湖麻鸭60羽,随机均分为试验组和对照组。试验组鸭皮下接种1:5稀释的鸭副粘病毒WF00D株SPF鸡胚绒尿液0.5mL/羽,对照组鸭皮下注射等量灭菌生理盐水,并分别置于25℃隔离环境下饲养与观察。于接种后第4d、8d、12d、16d和第20d,两组均随机取4羽鸭进行剖检,观察各器官的大体病变,同时,观察病理组织学变化。结果显示:试验组鸭除个体发育呈明显差异外,内脏器官在观察期间均有较轻微的炎性水肿或出血;病理组织学变化主要为器官充血或出血、炎性细胞浸润、细胞颗粒变性;超微病理学变化主要为细胞器变性,胞浆局灶性坏死、线粒体空泡化、粗面内质网脱颗粒。研究表明其病理演变过程具有一定的规律性。本研究为鸭副粘病毒流行规律的探究及致病机理的深入研究提供了依据。     

人工感染鸭瘟的病理学研究

用从自然病例中分离鉴定的鸭瘟病毒-乐山株(DPV-LS株)人工感染四川麻鸭,成功复制出鸭瘟(DP),并对其病理组织学和宿主细胞超微结构变化及病毒的形态结构进行观察.试验组鸭出现高热、神萎、不食、腹泻,剖解见食道、小肠、泄殖腔及各实质器官出血;组织学变化表现为各脏器出血、组织细胞变性、坏死,血管内皮受损,纤维素样坏死.超微病理学变化:肝细胞、网状细胞、上皮细胞、心肌细胞等的胞浆内粗面内质网严重扩张、核糖体脱落、空泡化,线粒体肿胀、嵴紊乱或断裂甚至形成空洞;胞核染色质浓集于核的周围,核形不整、核膜扩张或消失,核仁消失.同时在心、肝、脾、食道、泄殖腔、小肠的细胞内观察到了处于不同发育阶段的病毒粒子,成熟病毒粒子具有疱疹病毒的典型形态结构,呈球形或近似球形,直径150～200 nm,位于核膜间隙及胞浆内.     

Post-epizootic surveys of waterfowl for duck plague (duck virus enteritis)

Surviving birds from nine duck plague outbreaks in urban and confined waterfowl were sampled for duck plague (DP) virus and DP antibody during 1979-86. Duck plague virus was found in combined oral and cloacal swabs of birds from three outbreaks, and DP-neutralizing antibody was demonstrated in some birds from all nine outbreaks. Greater prevalence of DP antibody and higher titers were found in survivors from confined populations than from free-flying urban populations. Free-flying waterfowl from within 52 km of four DP outbreak sites were also sampled; virus was not found in any birds, but DP antibody was found in urban waterfowl in the vicinity of an outbreak in Potterville, Michigan. No evidence of exposure to or shedding of DP virus in migratory waterfowl was found in two regions where DP appears enzootic in urban and confined waterfowl (Eastern Shore of Maryland and the vicinity of Sacramento, California).