鸭出血性卵巢炎病毒血症研究

doi:10.3864/j.issn.0578-1752.2014.23.021

摘要/Abstract

摘要： 【目的】阐明鸭出血性卵巢炎病毒（DHOV-HB株）感染鸭后的病毒血症，为深入了解鸭出血性卵巢炎的发病机理、诊断和疫苗研究提供依据。【方法】24只250日龄北京鸭芯片标记后分别经口感染100倍稀释的DHOV-HB株（3×10⁴ELD₅₀），并设空白对照组15只，同等条件下分别在隔离器内饲养，逐日观察10日，各组每日经翅静脉采集10只鸭血液（尽可能采集同一只鸭血液），用于病毒分离和抗体测定。将攻毒后1—10 d的血清样本经卵黄囊途径接种6日龄SPF鸡胚，每份血清以0.1 mL/胚的剂量接种5枚SPF鸡胚，鸡胚接种后置于37℃条件下继续孵化，每日定时照胚2次，及时收获并统计24—168 h之内死亡的鸡胚，死亡鸡胚胚体经剪碎，研磨，离心后采用RT-PCR的方法检测病毒核酸，有1枚及1枚以上死亡鸡胚，且DHOV病毒核酸检测阳性，即将该鸭判为病毒分离阳性。同时采用中和试验测定攻毒后4—10 d的血清抗体，每个血清稀释度接种5枚SPF鸡胚。鸡胚接种途径及特异性死亡的判定方法同上。判定标准如下：血清原液保护80%（4/5）以上的鸡胚判为抗体阳性，保护20%—60%（1/5—3/5）的鸡胚判为抗体可疑，不保护鸡胚（0/5）判为抗体阴性。【结果】试验鸭感染DHOV后的病毒血症和免疫反应密切相关。感染后1—3 d，病毒血症出现并达到高峰，病毒分离均100%（10/10）阳性；4—6 d病毒分离率开始下降，分别为90%（9/10）、70%(7/10)和30%（3/10）阳性；7—10日血清中不再存在病毒，均为阴性(0/10)。中和试验结果表明攻毒后4 d，血清中病毒分离率下降的同时血清中即可能存在较低滴度的抗体，鸡胚死亡时间延后，此时抗体100%（10/10）阴性；攻毒后6 d的血清抗体80%（8/10）阴性，20%（2/10）可疑；攻毒后7 d 70%（7/10）阳性，10%（1/10）阴性，20%（2/10）可疑；攻毒后9 d 90%（9/10）阳性，10%（1/10）可疑；攻毒后10 d 100%（10/10）阳性。空白对照组血清病毒分离（1—10 d）和抗体检测（4—10 d）均为阴性。【结论】（1）DHOV口服感染成年鸭后，1—3 d日血清病毒分离率最高，感染后4 d 病毒分离率开始下降，感染后7 d血清病毒分离阴性。感染后7 d抗体70%（7/10）阳性，感染后10 d 100%（10/10）阳性；（2）病毒血症的持续时间与特异性免疫应答密切相关。

关键词: 鸭, 鸭出血性卵巢炎, 坦布苏病毒, 病毒血症

Abstract: 【Objective】 The objective of the study is to illustrate the viremia of ducks challenged with duck hemorrhagic ovaritis virus (DHOV-HB Strain) , to gain insight into the pathogenesis of DHOV and provide data for diagnosis and vaccine development.【Method】A group of twenty-four 250-day-old Peking ducks with implanted chip were infected with 100-fold diluted DHOV-HB (3×10⁴ELD₅₀) orally, 15 ducks as negative control were kept in isolation unit under the same condition, clinical signs were observed daily. On 1-10 day post inoculation (DPI), 10 serum samples were collected from each group via wing vein daily for virus-isolation and antibody determination (select the same duck as possible). The serum samples of 1-10 DPI were inoculated into 6-day-old SPF chicken embryos via yolk-sac route. Five SPF chicken embryos were inoculated for each sample at the inoculum of 0.1 mL per embryo. Then they were hatched at 37℃ further. The chicken embryos were observed regularly twice a day. Reap and count the death chicken embryos during 24-168 h in time. Then the death chicken embryos were cut into small pieces, and then centrifuged the grinding homogenate. The viral nucleic acid was detected by RT-PCR in the supernatant. If there are more than one (including one) death chicken embryos, and the nucleic acid testing was positive, then it was concluded that virus isolation was positive. Using the classical neutralization test method to detect the serum antibody of 4-10 DPI, 5 SPF chicken embryos were inoculated for each serum dilution. The inoculation method and criteria of embryos’ specific death were performed as previously described. The determination standards are as follows: results with serum protect more than 80% (4/5) chicken embryos, the serum antibody was recognized as positive, 20%-60%(1/5-3/5) were as doubtful, and 0%(0/5) as negative.【Result】The viremia and antibody responses were closely related after infection with DHOV. The viremia could be detected as early as 1 DPI, peaked at 1-3 DPI. By this time, the positive isolation rate of serum were all 100% (10/10). On 4-6 DPI, the positive isolation rate of serum began dropping which were 90% (9/10), 70% (7/10), and 30% (3/10), respectively, and no virus was recovered anymore on 7-10 DPI. The results of the neutralization test indicated that there were low titer antibodies in the serum while the virus isolation positive rate showed a decreasing trend on 4 DPI, showed up as the death time of the embryos postponed while the antibodies of the inoculated ducks were all negative temporarily. It was 80%(8/10) negative, 20%（2/10） doubtful on 6 DPI , 70% (7/10) positive, 10% (1/10) negative, 20% (2/10) doubtful on 7 DPI, 90% (9/10) positive 10% (1/10) doubtful on 9 DPI, and the antibodies of the inoculated ducks were all positive on 10 DPI. Virus isolation (1-10d) and antibody test (4-10d) results were all negative in the negative control ducks. 【Conclusion】The duration of viremia stage of ducks infected with DHOV-HB was short, the virus was recovered in 100% (10/10) on 1-3 DPI, decreased on 4 DPI and disappeared on 7 DPI. The antibody-positive rate was 70% (7/10) on 7 DPI and reached 100% (10/10) on 10 DPI. Further analysis showed that the quickly absence of viremia was closely related to specific antibody response. The antibody-positive rate was 70% (7/10) on 7 DPI and reached 100% (10/10) on 10 DPI.

Key words: ducks, duck hemorrhagic ovaritis, Tembusu virus, viremia

谢佳，韩春华，林健，段会娟，杨志远，赵际成，潘洁，刘月焕. 鸭出血性卵巢炎病毒血症研究[J]. 中国农业科学, 2014, 47(23): 4763-4768.

XIE Jia, HAN Chun-hua, LIN Jian, DUAN Hui-juan, YANG Zhi-yuan, ZHAO Ji-cheng, PAN Jie, LIU Yue-huan. Study on Viremia of the Ducks Infected with DHOV-HB Strain[J]. Scientia Agricultura Sinica, 2014, 47(23): 4763-4768.

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