Apoptosis induced in vivo by new type gosling viral enteritis virus

In this study, apoptosis was induced by new type gosling viral enteritis virus (NGVEV) in experimentally infected goslings is reported in detail for the first time. After 3-day-old goslings were orally inoculated with a NGVEV-CN strain suspension, the time course of NGVEV effects on apoptotic morphological changes of the internal tissues was evaluated. These changes were observed by histological analysis with light microscopy and ultrastructural analysis with transmission electron microscopy. DNA fragmentation was assessed with a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and DNA ladder analysis. A series of characteristic apoptotic morphological changes including chromatin condensation and margination, cytoplasmic shrinkage, plasma membrane blebbing, and formation of apoptotic bodies were noted. Apoptosis was readily observed in the lymphoid and gastrointestinal organs, and sporadically occurred in other organs after 3 days post-infection (PI). The presence and quantity of TUNEL-positive cells increased with infection time until 9 days PI. DNA extracted from the NGVEV-infected gosling cells displayed characteristic 180~200 bp ladders. Apoptotic cells were ubiquitously distributed, especially among lymphocytes, macrophages, monocytes, and epithelial and intestinal cells. Necrosis was subsequently detected during the late NGVEV-infection phase, which was characterized by cell swelling, plasma membrane collapse, and rapidly lysis. Our results suggested that apoptosis may play an important role in the pathogenesis of NGVE disease.     

雏鹅新型病毒性肠炎病毒对鸭胚成纤维细胞的适应性及增殖特性

为了获得适宜雏鹅新型病毒性肠炎病毒（NGVEV）体外培养的细胞系，开展了NGVEV强毒株在鸭胚成纤维细胞（DEF）适应性和增殖特性的研究。结果表明，NGVEV强毒株经尿囊腔接种10日龄鸭胚传4代，取尿囊液接种DEF，第1代无明显细胞病变；第2代培养至72～96hDEF出现颗粒状缺失、脱落，但无典型蚀斑出现；第3代培养至96～120h，DEF形成大小不等、圆形或椭圆形的典型蚀斑；第5代以后的NGVEV可稳定适应于DEF，典型细胞病变出现于48～72h。取适应DEF的NGVEV感染DEF细胞后制作超薄切片经电镜观察，可见典型的腺病毒粒子，大小为70～90nm。NGVEV在DEF上的TCID5。值随着传代次数增加而增高，由第1代的10^1.23增加到第8代的10^8.02，最高毒价出现于96～144h，96～120h是收获病毒的最佳时间。     

Quantitative analysis of virulent duck enteritis virus loads in experimentally infected ducklings

To better understand the pathogenesis of duck virus enteritis (DVE), the levels of viral DNA in various tissues of ducklings during acute stage of virulent duck enteritis virus (DEV) infection were investigated by using quantitative real-time polymerase chain reaction. The results show that the viral levels of DEV in systemic organs have a close correlation with the progression of disease. The rapid dissemination and active replication of virulent DEV in multiple systemic organs at the early phase of acute infection accelerate the progression of disease. The levels of viral DNA increase sharply soon after developed clinical signs of disease, and the extent of increase and the magnitude of DEV DNA load in various tissues of ducklings after the exhibition of clinical signs may be a critical determinant of the outcome of DEV infection. The relatively high levels of DEV in bursa and small intestine tissues of dead ducklings most likely reflect the abundance of target epithelial and lymphoid cells in these tissues, which therefore play a key role in the pathogenesis of acute DVE and manifest as severe tissue lesions on the bursa and small intestine.     

Immunohistochemical detection and localization of new type gosling viral enteritis virus in paraformaldehyde-fixed paraffin-embedded tissue

To determine the distribution and localization of new type gosling viral enteritis virus (NGVEV) in paraformaldehyde-fixed paraffin-embedded tissues of experimentally infected goslings, for the first time, an immunohistochemical (IHC) staining method was reported. Anti-NGVEV polyclonal serum was obtained from the rabbits immunized with purified NGVEV antigen, which was extracted by caprylic-ammonium sulphate method and purified through High-Q columns anion exchange chromatography. Three-day-old NGVEV-free goslings were orally inoculated with NGVEV-CN strain suspension as infection group and phosphate buffered saline solution (PBS) as control group, respectively. The tissues were collected at sequential time points between 0.5 and 720 h post inoculation (PI), and prepared for IHC staining and ultra-structural observation. The positive immunoreactivity could be readily detected in the lymphoid and gastrointestinal organs of infected goslings as early as 48 h PI, in the liver, kidney, pancreas and myocardium from 72 h, and in the cerebrum and cerebellum from 96 h, while it was hardly detected in the respiratory organs at any time. The positive staining reaction could be detected in NGVEV-infected goslings until 600 h PI, and no positive staining cell could be observed in the controls. The highest levels of viral antigen were found in the bursa of Fabricius (BF), thymus, proventriculus, gizzard and intestine tract, moreover, the liver, kidney, spleen, myocardium and pancreas were intensively and widely stained. The target cells had a ubiquitous distribution, especially included the epithelial cells, endothelial cells, superficial and crypt mucosal cells, glandular cells, fibrocytes, macrophages and lymphocytes, which served as the principal sites for antigen localization. The ultra-structural observation by transmission electron microscope (TEM) further indicated that NGVEV particles could be widely detected in the lymphoid and digestive organs of infected goslings from 72 h PI onwards. This work may be useful not only for offering a possibility of routine diagnosis of NGVE, but also for better understanding of the pathogenesis of the disease.     

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.     

Intestinal mucosal immune response against virulent duck enteritis virus infection in ducklings

Duck virus enteritis (DVE) is an acute and contagious herpes virus infection of duck, geese and swans with high morbidity and mortality. The development of specific mucosal immune system against duck enteritis virus (DEV) infection for ducks has been hindered by a lack of knowledge concerning the purification of immunoglobulin A (IgA) of duck. In the present work, the method for purification of duck immunoglobulin A was developed, and the induction of intestinal mucosal immune responses against DEV was studied by orally infected ducklings with virulent DEV. The results showed that a continuous increased DEV DNA levels were observed in blood and various organs examined of orally infected ducklings throughout the infection, which was accompanied by the development of infection in ducklings from mild progressed to severe pathological lesions. Furthermore, a marked increased level of DEV-specific IgA and IgG antibodies in bile, serum and the intestinal tract, as well as the density of IgA⁺ cells in intestine were detected between 1 and 12 days p.i., followed by a drastic reduction of the antibody levels and the density of IgA⁺ cells at 15 days p.i. The results indicate that the DVE infection can stimulate both IgA-dominated antibody immune responses in the intestinal tract, and IgG-dominated antibody systemic immunity in the serum of ducklings orally inoculated with virulent DEV. The severe lesions of the villus epithelial cells and the lymphoid organs can suppress the intestinal mucosal immune responses.     

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).     

Pathogenicity of a low-virulence duck virus enteritis isolate with apparent immunosuppressive ability

Duck enteritis virus (DEV) was isolated from commercial 2-to-6-wk-old white Pekin ducks experiencing 25%-30% mortality and high morbidity. Secondary infections with Pasteurella multocida, Riemerella anatipestifer, and Escherichia coli were frequently seen in affected ducks. The isolated virus was identical to the prototype DEV by virus neutralization test but differed from the classic DEV by causing lymphoid organ atrophy and inconsistent hemorrhagic lesions in the intestinal annular bands. Attempts to reproduce the disease in white Pekin ducks were unsuccessful until the virulence of the virus was increased by three passages in Muscovy ducklings. Significant thymic atrophy (P < or = 0.001) was detected during the first 10 days postinfection (DPI), but thymus size returned to normal by 17-24 DPI. However, bursal atrophy increased significantly (P < or = 0.001) from 4 DPI until the end of the experiment (39 DPI). Reduction in body weight was significant (P < or = 0.05) between 4 and 6 DPI. There was massive depletion of thymic and bursal lymphocytes with lymphoid necrosis in the thymus, bursa, spleen, and Harderian gland. Eosinophilic intranuclear inclusions were observed in thymus, bursa, spleen, esophagus, cloaca, liver, conjunctiva, and Harderian gland. Occasional intracytoplasmic inclusions were also found scattered in the epithelial cells of conjunctiva, esophagus, bursa of Fabricius, and cloaca. Virus was recovered from experimentally infected ducks from thymus, bursa, spleen, liver, kidneys, trigeminal ganglion, and cloaca during the first 10 days of infection. These findings suggest that a low-virulent DEV can cause a massive lymphoid atrophy and can sustain immunosuppression as noted by the secondary bacterial infection.     

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.     

复方中药“禽康散”对鸭瘟病毒感染鸭胚及成纤维细胞的保护效应

将不同浓度复方中药“禽康散”和鸭瘟病毒(Duck plague virus,DPV)分别以不同方式接种到鸭胚尿囊膜后观察其对DPV感染的鸭胚保护率;用MTT法测定“禽康散”对鸭胚成纤维细胞(DEF)的安全质量浓度和增殖的影响,及安全质量浓度范围内的“禽康散”对DPV感染鸭胚成纤维细胞(DEF)能力的影响.结果显示,“禽康散”在10日龄鸭胚内对DPV具有显著的杀灭和阻断感染的作用.“禽康散”对DEF的最大安全质量浓度为2-4 g/mL,在2-4～2-8 g/mL质量浓度范围内对DEF有显著的增殖和抑制DPV感染DEF的能力,且与加药方式和药物质量浓度有一定关系.结果表明,一定质量浓度的“禽康散”对DEF具有增殖作用,并对DPV在鸭胚内和细胞水平上均有较好的杀灭和拮抗作用.     

鸭肿头出血症病毒在鸭胚原代成纤维细胞中增殖特性的研究

将鸭肿头出血症病毒（DSHDV）强毒株经尿囊腔接种10日龄鸭胚，传3代，取第3代尿囊液接种鸭胚成纤维细胞（DEF），传代培养至有细胞病变出现，并对接毒细胞进行TCID50动态检测和透射电镜观察。结果显示，第1代接毒细胞培养至120h出现变圆、脱落，并形成少量蚀斑，第2代培养至96～120h，DEF形成大量圆形或椭圆形的典型蚀斑，第3代培养至72～96h，DEF出现典型蚀斑；此后可稳定适应于DEF，典型细胞病变出现于72～96h。DSHDV在DEF上的TCID50值随传代次数的增加而增高，由第1代的10^2.72增加到第10代的10^6.82，最高毒价出现于96h，该点是收获病毒的最佳时间。经透射电镜观察，可见DSHDV粒子呈球形或椭圆形，大小为60～75nm，无囊膜，具有双层衣壳。结果证实，DSHDV强毒株已经适应了DEF，传代后可获得较高的病毒效价。     

鸭肠炎病毒gL蛋白在鸡胚成纤维细胞中动态表达的检测

为研究鸭肠炎病毒(DEV)gL蛋白在感染鸡胚成纤维细胞(CEF)过程中的表达情况,本研究以DEVClone-03基因组为模板,应用PCR方法分别扩增得到截短的(gLt,181 bp～711 bp)和全长的gL(1 bp～711 bp)两个基因片段。将gLt基因片段克隆至pET-30a原核表达载体,转化E.coli BL21(DE3),经IPTG诱导表达并对表达产物进行纯化复性,免疫BALB/c小鼠,制备鼠抗gL蛋白多克隆抗体。同时将全长gL基因克隆至真核表达载体pcDNA3.1(+),构建真核表达重组质粒pcDNA-gL,转染293T细胞。采用获得的抗gL蛋白抗体检测DEV感染CEF后及真核表达质粒pcDNA-gL转染293T细胞后gL蛋白在不同时间点的表达情况。结果表明,在pcDNA-gL转染293T细胞后12 h应用western blot方法能够检测到gL蛋白的表达,其表达量随着转染时间增加而增加;在病毒感染CEF后24 h应用间接免疫荧光方法能够检测到gL蛋白少量的表达,western blot方法在病毒感染CEF48 h后检测到gL蛋白的表达,其表达量随着病毒感染时间增加而增加。上述结果提示,编码gL蛋白基因可能是病毒复制的晚期表达基因。     

I型鸭肝炎病毒在鸭胚成纤维细胞系中的增殖特性研究

为研究I型鸭肝炎病毒(DHV-1)在鸭胚成纤维细胞系(DEF-h)中的增殖规律,本研究将DHV-1野毒株(ZJ-08株)接种DEF-h细胞,连续传代3次后,观察病毒对细胞的致病变效应(CPE),并对病毒核酸及其蛋白的表达和病毒增殖特性进行了鉴定。研究结果显示,DHV ZJ-08株能够在DEF-h中有效增殖,并产生典型的CPE,病毒蛋白在细胞中也获得了良好表达,并与DHV-1特异性抗体发生反应。一步生长曲线显示ZJ-08株病毒感染DEF-h 12 h开始增殖,36 h～60 h进入快速增殖期,72 h达到峰值,TCID50为10-4.3/mL。本实验为进一步的DHV-1的致病机理和细胞灭活疫苗的研制奠定了基础。     

An outbreak of duck viral enteritis (duck plague) in domestic Muscovy ducks (Cairina moschata domesticus) in Illinois.

Duck viral enteritis (DVE) was diagnosed in an outbreak of the disease in a resident population of Muscovy ducks (Cairina moschata domesticus) on a privately owned multispecies game bird production facility in Illinois, where it claimed 625 ducks. This disease condition had not been reported previously in domestic ducks in Illinois. Although other varieties and age groups of domestic waterfowl (i.e., black ducks, rhumen ducks, Pekin ducks, ducklings, and geese) were present on the game bird farm, the morbidity and mortality (100%) in this epornitic was solely limited to adult ducks of the Muscovy lineage. The clinical signs in the affected ducks were lethargy, diarrhea, dehydration, and death within 2-3 hr of onset of symptoms. Gross pathologic changes were nonspecific and included ecchymotic hemorrhage, effusion of fluid and blood within body cavities reflective of an acute systemic infectious disease. Light microscopic findings were necrosis of primarily digestive lining epithelium and variable lymphohistiocytic infiltration within mucosal and serosal connective tissues. Intranuclear inclusions resembling characteristic herpetic (i.e., Cowdry type A) inclusions were observed primarily in the digestive, respiratory, and reproductive tracts; liver; and spleen. Esophageal candidiasis, bacteriosis, and systemic Pasteurella anatipestifer infections, thought to be concurrent or opportunistic infections, were present in several ducks. DVE virus was demonstrated in infected Muscovy duck embryo fibroblast cells by direct DVE virus-specific fluorescent antibody staining.     

The occurrence of virus enteritis (duck plague) in wild birds]

296 samples from wild birds of 15 species were incorporated into long term exploration of duck plague on ducks in farms. By using virological and serological standard methods 9 virus carriers and 20 serum samples showing positive antibody titers could be detected. The epidemiology as well as the relation of the incidence of duck plague in wild birds and farm poultry is discussed.