[1.8-1.11]细小病毒病原体诊断方法和病毒疫苗研制材料及方法
[第二章]新型鸭细小病毒疫苗的研究成果
[第四章-参考文献]鸭细小病毒灭活疫苗研制的讨论与结论
摘 要
2014 年11 月以来,我国部分地区所饲养的肉鸭发生了以雏鸭发育迟缓、上下喙萎缩、舌头外
伸为特征的疾病。根据其发病临床特征命名为鸭短喙-侏儒综合征(duckshort beak and dwarfism
syndrome,SBDS)。本实验室通过病原分离以及动物回归实验,确定了引起 SBDS 的病原为鸭
细小病毒(Duck Parvovirus)。目前,防控该病尚无商业化的疫苗,给我国养鸭业造成了较大
经济损失。鉴于此,作者选取鸭细小病毒流行株制备了鸭细小病毒病灭活疫苗,围绕新制品的
研制对其实验室试验进行了一些有益的探索。
将15 只1日龄的樱桃谷雏鸭随机分成三组:第1组(口服组),每只口服 1 m L 病毒尿囊液:
第2组(肌肉注射组),每只肌肉注射病毒尿囊液 1 m L(EID50 为10-4.5/0.2m L),第 3组
(对照组),每只雏鸭口服 1 m L 生理盐水。在感染鸭细小病毒后 7、14、21 d 分别对三组动
物称重、测量喙长,口服组雏鸭体重极显着低于对照组,肌注组低于对照组,因此确定最佳感
染途径为口服途径。
为探明该病毒的生物学特性,将SD 株鸭细小病毒用鸭胚传至40 代,分别用10、20、30、40
代病毒对动物进行攻毒实验。结果表明,F10 代鸭细小病毒组雏鸭体重和喙长和对照组差异极
显着,感染F20?
F30 代病毒组雏鸭体重和喙长与对照组对比差异显着,感染病毒 F40 代病毒组雏鸭和对照组差
异不显着,感染后,21 d 均可以检测到排毒。综上所述,该病毒传至40 代,病毒对动物的致
病力降低。将保存的鸭细小病毒分离株经鸭胚传代五次,收获病毒尿囊液。
9000 r/min 离心10 min.取上清,加双抗。将提前处理好的尿囊液和吐温-80 按比例制作水相,
按照油相和水相 2:1 比例将二者混匀,制备油乳剂灭活疫苗。并对种毒纯净性和疫苗的外观、
粘度、稳定型、安全性和保存期的质量检验。为评价制备的灭活疫苗的免疫效力,将50 只1
日龄的雏鸭随机分为 5组,每组 10 只,1?
4组为免疫组,免疫剂量分别为100μL、250μL、500μL、1000μL, 第5组为对照组。免疫后 7 d
进行免疫保护实验,观察实验鸭的临床症状和排毒情况。结果表明疫苗外观为乳白色、油包水
型、无外源病毒污染、粘度符合标准、稳定性良好、对动物无不良影响、4℃可保存 1年以
上;对疫苗安全性检验用20 只1日龄雏鸭,随机分为两组,每组 10 只。一组注射成品疫苗为
疫苗组,二组注射白油佐剂为对照组。疫苗组和对照组对比后精神状态正常、注射部位没有发
生炎症,证明安全性检验合格;免疫保护实验中,对照组在攻毒后 3d 泄殖腔肛拭子检测到部
分阳性,100μl?1000μL 四组免疫组检测结果全部阴性。攻毒后 5 d 对照组全部阳性,而1?4 组
免疫组检测结果全部阴性。直到攻毒 21 d 后,对照组检测仍然全部呈阳性,四组免疫组检测全
部阴性。上述结果表明制备的鸭细小病毒灭活疫苗安全、稳定、易于储存运输,雏鸭免疫后可
以获得坚强保护。
本研究通过不同代次病毒接种动物,探明该病毒的生物学特性。为了有效预防本病的蔓延,用
该毒株制备鸭细小病毒灭活疫苗,疫苗安全有效,能在雏鸭易感日龄提供保护,为鸭细小病毒
疫苗的研制提供了依据。
关键词:鸭细小病毒;鸭短喙-侏儒综合征;灭活疫苗;最小免疫剂量 Abstract
Since November 2014, ducks reared in parts of China have developed diseasescharacterized by stunted
growth of ducklings, atrophy of the upper and lower jaw, andtongue extension. According to its
clinical characteristics, it was named duck short beakand dwarf syndrome (SBDS )。The
laboratory determined that the pathogen responsible forSBDS is duck parvovirus disease through
pathogen isolation and animal regressionexperiments. At present, there is no commercial vaccine for
the prevention and control ofthe disease, which has caused great economic losses to the domestic duck
industry. In viewof this, the duck duck parvovirus epidemic strain was used to prepare an
inactivatedvaccine against duck parvovirus disease, and some useful explorations of the
laboratoryexperiments were carried out around the development of new products.
Fifteen 1 d Cherry Valley ducklings were randomly divided into three groups: Group1 (oral
group ),each oral 1 m L of viral allantoic fluid: Group 2 (intramuscular injectiongroup)each
only intramuscular injection of 1 m L of virus allantoic fluid (EID50 10-4.5/0.2m L )and group 3
(control group )each duck was orally administered with 1 m L ofphysiological saline. Groups 1 to 2
were housed in the same animal house and the controlgroup was housed in another animal house.
After the duck parvovirus was infected, threegroups of animals were weighed and measured for beak
length on the 7th, 14th, and 21stdays. The data showed that the weight of the ducklings in the oral
group was significantlylower than that of the control group. The intramuscular injection group was
lower than thecontrol group, but not significantly, it can be determined that the optimal route of