Pathogen Identification and Tissue Histopathology on Plague Virus-infected Mule Ducks
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摘要:
目的 2020年8月，福建省福州地区某鸭场饲养的半番群发病，发病率约为65%，病死率高达90%以上，感染鸭皮肤表面可见大量出血，为了明确其病原和病理组织学特征。 方法 分别采集发病鸭的肝脏、脾脏、胰腺、肾脏、皮肤等组织进行鸭常见病原检测、病毒分离鉴定、基因序列测定分析及病理组织学观察。 结果 病原检测结果显示，所采集组织仅鸭瘟病毒PCR检测呈阳性，其余病原均为阴性；对阳性样品进行病毒分离鉴定，结果从发病鸭内脏组织和皮肤中分离出鸭瘟病毒，命名为FJ2020176。对新分离鸭瘟病毒FJ2020176株UL2基因进行序列测定与分析发现，其与鸭瘟强毒株CHv株、CV株和2085株的核苷酸同源性为97.8%～99%；与疫苗株VAC株、Attenuatedstrain 1株和Attenuated strain 2株等弱毒株相比，FJ2020176株在UL2基因中存在528 bp的核苷酸插入，表明该毒株为鸭瘟病毒强毒株。病理组织学观察显示，感染鸭可见肝脏局灶性坏死、出血；脾脏组织白髓减少，淋巴细胞坏死、脱落；肾脏出血、淤血；法氏囊出血，淋巴小结坏死；皮下出血。 结论 从皮肤大量出血的发病鸭中分离到1株鸭瘟病毒强毒株，表明皮肤出血亦为鸭瘟病例的临床特征，以上结果为临床上鸭瘟的诊断提供了新的试验数据。 Abstract:Objective The pathogen causing a viral disease with a 65% incident rate, 90% mortality, and massive skin hemorrhage on the birds at a mule duck farm in Fuzhou, Fujian in August 2020 was isolated with its pathogenic characteristics identified. Method The heart, liver, spleen, pancreas, kidney, skin, and other tissues of the diseased ducks were collected for the pathogen detection including virus isolation and identification, gene sequencing, and histopathological examinations. Result PCR showed positive results only on the collected tissues from the plague virus-infected ducks. Subsequent virus isolation and identification on those positive specimens were performed, and the virus code-named as FJ2020176. The UL2 gene of the strain was sequenced to show a 97.8-99.9% nucleotide homology with the virulent strain CHV, CV, and 2085. Compared to the vaccine strain VAC, the attenuated strain 1, and the attenuated strain 2, FJ2020176 had a 528bp nucleotide insertion in its UL2 gene confirming it to be a virulent strain of duck plague virus. The histopathological examination under a microscope showed the following observations in the infected ducks: necrosis and hemorrhage on the focal liver, decreased white pulp on the spleen tissue, necrotic and exfoliated lymphocytes, hemorrhage and blood stasis on the kidney, necrosis on the lymph nodes, and hemorrhage on the bursa of Fabricius, in addition to the exterior skin hemorrhage. Conclusion The virulent FJ2020176 was isolated from the diseased ducks with massive skin hemorrhage and severe infection and mortality rates. The bleeding on the skin was deemed a significant symptom for clinical diagnosis of the duck plague cases. -
Key words:
- mule duck /
- duck plague virus /
- isolation and identification /
- histopathology
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图 1 发病鸭鸭剖检病变图
注：A：头部；B：皮肤；C：肝脏。
Figure 1. Necropsy lesions in diseased ducks.
Note: A: head; B: skin; C: liver.
图 2 临床采集样品的PCR检测
注：M：DNA 2 000 Marker；1：阳性对照；2：阴性对照；3：鸭瘟病毒（肝脏）；4：鸭瘟病毒（脾脏）；5：鸭瘟病毒（心脏）；6：鸭瘟病毒（肾脏）；7：鸭瘟病毒（法氏囊）；8：鸭瘟病毒（皮肤）；9.禽流感病毒；10：新城疫病毒；11：鸭肝炎病毒；12：禽坦布苏病毒；13：水禽细小病毒；14：鸭3型腺病毒；15：禽4型腺病毒；16：呼肠孤病毒；17：鸭星状病毒；18：鸭圆环病毒。
Figure 2. PCR detection on clinical samples
Note: M: DNA 2000 marker; 1: positive control; 2: negative control; 3: duck plague virus on liver; 4: duck plague virus on spleen; 5: duck plague virus on heart; 6: duck plague virus on kidney; 7: duck plague virus on bursa of Fabricius; 8: duck plague virus on skin; 9: avian influenza virus; 10: Newcastle disease virus; 11: duck hepatitis virus; 12: avian Tembusu virus; 13: waterfowl parvovirus; 14: duck adenovirus 3; 15: avian adenovirus 4; 16: reovirus; 17: duck astrovirus; 18: duck circovirus.
图 3 临床采集样品的PCR检测
注：M：DNA 2 000 Marker；1：阳性对照；2：阴性对照；3：内脏样品鸭胚传代鸭瘟；4：皮肤样品鸭胚传代鸭瘟；5：禽流感病毒；6：新城疫病毒；7：鸭肝炎病毒；8：禽坦布苏病毒；9：水禽细小病毒；10：鸭3型腺病毒；11：禽4型腺病毒；12：呼肠孤病毒；13：鸭星状病毒；14：鸭圆环病毒。
Figure 3. PCR detection on clinical samples
Note: M: DNA 2000 marker; 1: positive control; 2: negative control; 3: duck plague passage from duck embryo in viscera samples; 4: duck plague passage from duck embryo in skin samples; 5: avian influenza virus; 6: Newcastle disease virus; 7: duck hepatitis virus; 8: avian Tambusu virus; 9: waterfowl parvovirus; 10: duck adenovirus 3; 11: avian adenovirus 4; 12: reovirus; 13: duck astrovirus; 14: duck circovirus.
图 4 分离病毒的PCR扩增结果
注：M：DNA 2 000 Marker；1：阳性对照；2：阴性对照；3：FJ2020176。
Figure 4. PCR amplification result on isolated virus
Note: M: DNA 2000 marker; 1: positive control; 2: negative control; 3: FJ2020176.
图 6 感染鸭组织病理切片
注：A. 肝脏（×100）；B. 肝脏（×400）；C. 脾脏（×100）；D. 脾脏（×400）；E. 肾脏（×100）；F. 肾脏（×400）G. 法氏囊（×100）；H. 法氏囊（×400）；I. 皮肤（×100）；J. 皮肤（×400）。
Figure 6. Pathological slices of infected duck tissues
Note: A: liver (×100). B: liver (×400). C: spleen (×100). D: spleen (×400). E: kidney (×100). F: kidney (×400). G: bursa of fabricius (×100). H: bursa of fabricius (×400). I: skin (×100). J: skin (×400).
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