一株以肝脾白色坏死点为特征的鹅源番鸭呼肠孤病毒的分离鉴定

郑欣; 胥焯然; 宫晓; 江丹丹; 程晓霞; 朱小丽; 郑敏; 曾丽; 肖世峰; 曾显成; 陈少莺; 陈仕龙

一株以肝脾白色坏死点为特征的鹅源番鸭呼肠孤病毒的分离鉴定

Isolation and Identification of a Goose-origin Muscovy Duck Reovirus Causing White Pinhead Necrotic Foci on Liver and Spleen in Geese

摘要

摘要:
目的通过对雏鹅以肝脾白色坏死点为特征的病料进行病原分离与鉴定，为该病的防控提供科学依据。
方法利用PCR检测排查病原，挑选阳性病料接种番鸭胚和番鸭成纤维细胞（muscovy duck embryo fibroblast cells, MDEF）进行病毒分离，对分离毒再进行RT-PCR鉴定、病毒关键基因序列分析、动物回归试验验证。
结果成功分离出一株鹅源番鸭呼肠孤病毒（goose-origin muscovy reovirus, Go-MDRV），命名为JS2022株。分离毒接种番鸭胚至第6代时，番鸭胚死亡时间稳定在3～5 d，胚体出血，肝脏有大小不一的出血点和坏死灶，发育受阻。将分离毒尿囊液接种MDEF，出现细胞圆缩、崩解等病变。将JS2022株σB和σC基因的核苷酸序列与鹅源番鸭呼肠孤病毒、番鸭呼肠弧病毒（muscovy duck reovirus, MDRV）、新型鸭呼肠孤病毒（novel duck reovirus, NDRV）和禽呼肠孤病毒（avian reovirus, ARV）等毒株序列进行同源性比对，结果显示JS2022株与鹅源番鸭呼肠孤病毒毒株的同源性最高，核苷酸同源性范围分别为99.1%～99.5%和99.3%～99.9%；动物回归试验成功复制出与临床发病鹅相同的病症。
结论本试验成功从病死鹅的肝脏和脾脏中分离鉴定1株鹅源番鸭呼肠孤病毒 JS2022株，为鹅源番鸭呼肠孤病毒病的防治提供参考。

Abstract:
Objective Pathogen of the disease characterized by white pinhead necrotic foci on the liver and spleen in geese was isolated and identified.
Methods PCR was used to screen possible pathogens from tissue specimens of diseased birds. Subsequently, the suspect pathogens were inoculated in Muscovy duck embryos and Muscovy duck embryo fibroblasts (MDEF) cells to identify positive isolate for further confirmation by RT-PCR, genes sequencing, and a challenge test on gooselings.
Results A strain of goose-origin Muscovy duck reovirus (Go-MDRV), JS2022, was obtained and inoculated into Muscovy duck embryos causing stunted growth. After 6 viarl passages, mortality of the infected embryos stabilized to occur in 3-5 d post-inoculation. Haemorrhagic spots and the typical pinhead necrotic foci were visible on the liver of the dead embryos. On MDEF cells, the inoculation induced cytopathic cell shrinkage and disintegration. By comparing the nucleotides of σB and σC of JS2022 with those of Go-MDRV, Muscovy duck reovirus (MDRV), novel duck reovirus (NDRV), and avian reovirus (ARV), the greatest homology as found between JS2022 and Go-MDRV with the ranges of 99.1-99.5% on σB and 99.3-99.9% onσC. In the challenge test, the infected gooselings had symptoms identical to those shown in the clinical cases.
Conclusion A strain of Go-MDRV isolated from the diseased geese bearing the specific symptom of white necrotic foci on the liver and spleen was identified and code-named JS2022.

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