Humoral immune response of the duck to duck hepatitis virus: virus-neutralizing vs. virus-precipitating antibodies

Ducks were induced to develop high-level duck hepatitis virus (DHV)-neutralizing antibodies by inculation with a chicken-embryo-adapted DHV via subcutaneous, intramuscular, and intratracheal routes. Administration of the DHV orally in a gelatin capsule failed to stimulate immune response in the ducks. Contact controls of these ducks also remained negative for anti-DHV antibodies. These observations indicated that the DHV administered orally, in gelatin capsule, failed to infect the ducks. None of numerous duck anti-DHV immune sera, with virus-neutralizing activity in the range of 1.8 to 5.57 log10 median- embryo-infective-dose (EID50) neutralization index, developed precipitin lines against a variety of DHV preparations tested in low- and high-ionic-strength agar. The results suggest that the agar-gel immunodiffusion test is unsuitable for serologic testing of duck sera for anti-DHV antibody activity. Virus-neutralizing activity was revealed in both immunoglobulin M (IgM) and IgG classes of sera of actively immunized ducks. Immunodiffusion tests of Sephadex G-200 fractions of 1-day-old duckling sera with monospecific rabbit anti-duck IgM (DIgM) serum failed to detect DIgM. These results demonstrated that the IgM is not being transferred from the dam to the newly hatched ducklings. Seven- and 14-day-old ducks had DIgM in their sera. However, this IgM had no DHV-neutralizing activity, indicating that it was newly developed by the ducklings, which had no active DHV immune response, not having been exposed to DHV.     

一种“新型鸭瘟”的报道

鸭瘟是由疱疹病毒科的鸭瘟病毒引起的一种急性败血性传染病,是危害养鸭业的二类传染病之一.前几年,很少爆发大型鸭瘟,而且及时的预防措施一般能防止鸭瘟的流行和发生.2000年以来,一种新型鸭瘟已在我省的几个地区流行,且呈爆发性,其临床症状及剖检变化与传统鸭瘟相同,但该病主要侵害1月龄以下的雏鸭,成年鸭很少发病,常规的鸭瘟疫苗不能预防该病的发生,且发病后用鸭瘟抗血清治疗无效.根据对这几个地区的流行病学调查,该病的发病率可达70%～100%,死亡率高达50%～80%.由于目前对该病无有效的防治措施,因而给我省的养鸭业造成了重大的经济损失.现就这一“新型鸭瘟”(暂命名)的临床症状、剖检变化、病原分离与培养、电镜观察及国内研究进展作一综述.     

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).     

Comparison of the kinetics of the specific cellular immune response to duck hepatitis B virus in infected and immune ducks.

The kinetics of the cell mediated immune response by ducks acutely and chronically infected with, or immune to infection by duck hepatitis B virus (DHBV) was determined. This was measured by an antigen specific blastogenesis assay to duck hepatitis B surface antigen (DHBsAg) and duck hepatitis B core antigen (DHBcAg) using peripheral blood mononuclear cells (PBMC). The three outcomes of acute infection by DHBV were either clearance from both serum and liver, clearance from serum but not liver, or the development of persistent viraemia. Acutely infected ducks that failed to clear the infection also failed to develop a significant cellular immune response to both antigens. Ducks with chronic infection acquired as neonates or as the result of the failure to clear acute infection had an increasing cellular immune response over time. Two groups of immune ducks were examined. These were either ducks that had become immune following infection or that had been vaccinated. Both groups of ducks demonstrated significant cellular responses following challenge with DHBV irrespective of the level of their responses before challenge. However, there was a reduction in the response of their PBMC over a 4-week-period postchallenge. The range of cellular immune responses to DHBV antigens observed in this study has a number of counterparts in hepatitis B infection of humans. Coupled with the defined clinical outcomes that can be established in the duck/DHBV model, further study of the cellular immune response to DHBV is warranted.     

鸭瘟病理组织学动态观察

鸭瘟病毒（DPV）强毒经人工感染和同居感染成年鸭后，采用聚合酶链反应（PCR）检测为鸭瘟后，在不同时间段对各给织器官的病理组织损伤进行了观察．表现为：人工接种后24h被检器官组织显现出病理变化．机体中枢免疫器官法氏囊、胸腺表现为淋巴细胞数量降低，组织间隙加大；脾脏组织病变较为严重，其余器官组织均出现程度较轻的组织损伤。接种后48h，中枢免疫器官的淋巴细胞极度减少、网状细胞增生、器官组织结构模糊不清，充血、出血严重；一些生命重要器官则出现明显细胞肿胀，肠道等器官组织也有细胞变性、出血等不可逆病理变化。居感染组织损伤与人工接种组织相似，只是发生时间偏后约50h。提示：接种DPV强毒的感染鸭和同居鸭的免疫器官严重受损，甚至引超免疫抑制。此外，两组实验鸭的肝细胞、肾小管上皮细胞及脾脏、法氏囊、胸腺的网关细胞中的核内包涵体结构，可在病理组织学上为鸭瘟诊提供依据。     

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…     

Laboratory confirmed outbreaks of duck virus enteritis (duck plague) in the United Kingdom from 1977 to 1982

From 1977 to 1983 the Central Veterinary Laboratory, Weybridge confirmed 19 outbreaks of duck virus enteritis in the United Kingdom. All the outbreaks involved collections of captive waterfowl and there were no reported cases in commercial ducks. In many instances the disease was associated with contact with migrating waterfowl, particularly male mallards (Anas platyrhynchos). Muscovy ducks (Cairina moschata) and related species appeared to be particularly susceptible. The most sensitive system for isolating the virus was muscovy duck embryo tissue cultures. The duckling inoculation test was found to be the most reliable method of confirming the disease.     

鸭瘟的诊断与免疫防治

鸭瘟又名鸭病毒性肠炎（DVE）,是鸭、鹅的一种疱疹病毒感染的急性、接触性传染病。其特征是侵害血管、组织出血、消化道黏膜糜烂、淋巴器官出现病变、实质器官退行性病变。临床主要表现为腿软、走动困难、不愿下水、肿头,故名“大头瘟”,是危害养鸭业的三大疾病之一。     

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.     

PCR用于鸭瘟病毒诊断的研究

根据鸭瘟病毒UL6和UL7基因序列,设计合成了一对引物,以2株疫苗株、1株强毒株和1株山东分离株DNA为模板,进行PCR扩增,得到预期690bp的目的片段.将扩增的目的片段克隆到pMD18-T载体,经Amp平板筛选,HindⅢ、BamHⅠ双酶切鉴定,获得阳性重组质粒.对重组质粒进行序列测定,与参考序列比较,山东分离株与参考序列的同源性为99.7%,其余3株DPV与参考序列的同源性均为100%.应用PCR可检测人工感染和自然感染鸭瘟的组织中的鸭瘟病毒,表明PCR检测鸭瘟病毒具有很高的特异性、敏感性,该法能够用于鸭瘟急性及亚临床感染的检测与诊断.     

应用ELISA检测鸭瘟抗体的研究

将粗提鸭瘟病毒经超速离心后作为包被抗原，以健康鸭 IgG 提纯后免疫兔，制备兔抗鸭 IgG，并用改良过碘酸钠法进行辣根过氧化物酶标记，建立了检测鸭瘟病毒抗体的间接酶联免疫吸附试验(ELISA)，经对不同血清样品的检测，表明此方法特异性高，操作简便，适于大批鸭群抗体水平监测和流行病学调查，为合理免疫和科学预防提供了技术手段。     

Identification of duck plague virus by polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed for detecting duck plague virus. A 765-bp EcoRI fragment cloned from the genome of the duck plague vaccine (DP-VAC) virus was sequenced for PCR primer development. The fragment sequence was found by GenBank alignment searches to be similar to the 3' ends of an undefined open reading frame and the gene for DNA polymerase protein in other herpesviruses. Three of four primers sets were found to be specific for the DP-VAC virus and 100% (7/7) of field isolates but did not amplify DNA from inclusion body disease of cranes virus. The specificity of one primer set was tested with genome templates from other avian herpesviruses, including those from a golden eagle, bald eagle, great horned owl, snowy owl, peregrine falcon, prairie falcon, pigeon, psittacine, and chicken (infectious laryngotracheitis), but amplicons were not produced. Hence, this PCR test is highly specific for duck plague virus DNA. Two primer sets were able to detect 1 fg of DNA from the duck plague vaccine strain, equivalent to five genome copies. In addition, the ratio of tissue culture infectious doses to genome copies of duck plague vaccine virus from infected duck embryo cells was determined to be 1:100, making the PCR assay 20 times more sensitive than tissue culture for detecting duck plague virus. The speed, sensitivity, and specificity of this PCR provide a greatly improved diagnostic and research tool for studying the epizootiology of duck plague.     

The immune response of the chick following viral vaccinations and immunization with sheep red blood cells

The effects of viral vaccinations and immunization with sheep red blood cells (SRBC) on the humoral response of pullets were investigated. Pullets were vaccinated with Marek's disease virus, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and infectious bursal disease virus at appropriate ages used in commercial practice. At seven weeks, the pullets were intramuscularly immunized with SRBC. NDV and IBV antibodies were detected by hemagglutination-inhibition tests. Hemagglutination (HA) titers were established against SRBC. IBV antibody titers were not affected by vaccination or by immunization with SRBC. NDV antibody titers were significantly increased by vaccination and by immunization with SRBC. The SRBC agglutinin response was also positively affected by vaccination. The HA titer increase consisted of a rise in 2-mercaptoethanol (2-ME)-sensitive antibodies and a fall in 2-ME-resistant antibodies.     

Pathogenesis of digestive tract lesions in duck plague.

White Pekin ducklings were inoculated orally with duck plague virus. Tissues from the digestive tract were collected daily after inoculation and examined by light, electron and fluorescent microscopy. There were necrosis and degeneration of stratified squamous epithelium of the esophagus and cloaca, epithelium of intestinal crypt and esophageal submucosal glands, macrophages in the lamina propria, and submucosal fibrocytes and lymphocytes. Submucosal hemorrhages occurred after degeneration and necrosis of lymphocytes, macrophages, fibrocytes and epithelial cells. Viral antigens were detected in all these cells by use of fluorescein-labeled antibodies. With the electron microscope, nucleocapsids were seen in the nuclei, budding through the inner nuclear membrane; enveloped virions were present in cytoplasmic vacuoles of macrophages, epithelial cells and fibrocytes. In lymphocytes, nucleocapsids were also in the nuclei, but karyorrhexis and cytolysis occurred before viral maturation was completed.     

鸭瘟活疫苗生产工艺改进试验

利用尿囊腔接种法替代绒毛尿囊膜接种法生产鸭瘟活疫苗,收获的胚液和绒毛尿囊膜及胎儿的病毒含量(ELD50)均高于<中华人民共和国兽用生物制品规程>2000年版中的病毒含量标准,而且降低了早死率,提高了鸡胚利用率.