【作者】 刘建；

【导师】 苏敬良；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 本实验通过鸭胚尿囊腔接种，对我国几个省市采集的12份死于疑似鸭病毒性肝炎的病鸭肝组织进行病原分离，获得12个病毒分离株。这些分离株对10日龄鸭胚的致死率为100％，人工感染6日龄雏鸭的致死率为0-100％不等。死亡鸭呈角弓反张姿势，剖检可见肝脏肿大，有出血点或出血斑。将12株分离株经鸭胚传至第5代，收集鸭胚尿囊液毒测定这些毒株的ELD₅₀为10^-7.32／0.2ml-10^-3.5／0.2ml不等。用鸡抗1型DHV及新型DHV的血清和12株分离株进行中和试验，结果表明：有7株为新型DHV，有2株为1型DHV，还有3株无法确定其血清型。 通过对新型弱毒株（B株）进行鸭胚传代致弱后，稀释成不同倍数对1日龄雏鸭进行免疫，并于10日龄时用新型鸭肝炎强毒攻毒，观察雏鸭的死亡情况。由结果可知，B₂₀株在20倍稀释时，保护率达100％，而稀释到100倍时则完全失去保护作用。同时对免疫的雏鸭采血，中和试验测定血清的效价。免疫雏鸭在第10天时抗体水平达到高峰，然后呈逐渐下降趋势。结果表明，B株经过鸭胚传代致弱后在毒力下降的同时仍保留一定的免疫原性，可以作为疫苗的候选株。 此外，本实验用鸭胚肝细胞（DEL）培养了新型鸭肝炎病毒，并详细观察了鸭肝炎病毒引起的细胞病变，发现该病毒适于鸭胚肝细胞上培养。通过细胞免疫荧光染色，发现肝细胞本身自发荧光，不适于免疫荧光检测；采用感染新型鸭肝炎强毒后死亡鸭的肾细胞做滴片进行荧光染色，可检测到病毒抗原存在，为临床上快速诊断新型鸭病毒性肝炎提供一种检测方法。更多还原

【Abstract】 Twelve virus isolates were obtained through duck embryo allantoic sac inoculation from duckling liver samples collected from several provinces in China. All these ducklings were diagnosed clinically dying from duck virus hepatitis. The lethal rate of these isolates was 100 percent when inoculating 10-day-old duck embryos. When inoculating 6-day-old ducklings with these isolates, the mortality rate was among 0-100%. Dead ducklings showed the sign of opisthotonos, with swollen and punctuate hemorrhage in their livers. The ELD50 of these virus strains detected with duck embryos were among 10-7.32-10-3.5. Neutralizing test was done using standard serum against DHV type 1 serum and new serotype DHV. 7 isolates of them belonged to new serotype DHV, 2 belonged to DHV type 1, another 3 isolates were nontypable.A new serotype virus (B strain) was attenuated with duck embryos propagation and designated B20 strain.It was diluted into different concentration to immunize 1-day-old ducklings and the immunized ducklings were challenged with new serotype duck hepatitis virus in the 10th day. The result indicated that the protection rate of 20 fold dilution of B20 could reach to 100 percent. However, 100-fold dilution of B20 provided no protection effect. The serum of the immunized ducklings were collected and the antibody titer were measured by neutralizing test. The antibody level reached to the peak in 10 days old and then showed a downward trend. We can make the conclusion that B20 strain could provide protection aagainst the infection of new serotype DHV. It mey be used as the candidate vaccine strain.In addition, the new serotype DHV was inoculated to the duck embryo liver cell to culture, and the cytopathic effect was caused. The results showed that this virus was adapt to be cultured in liver cells of duck embryo. We found the liver cell culture were not suitable for the immunofluorescent detection for the cell emitted fluoresce spontaneous. Meanwhile, the kidney cell of ducklings died from experimentally infection with new serotype DHV was immunofluorescent.dyeing. The vival antigen could be detected by indirect immunofluorescent assay. We found the IFA could be used as rapid diagnostic technique for new serotype Duck Virus Hepatitis.更多还原