【作者】 张璐；

【导师】 刁有祥；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2015， 硕士

【摘要】 自2010年4月起,我国多地蛋鸭、种鹅养殖场相继爆发了一种以产蛋量严重下降为主要临床症状的新型传染病,经分离鉴定确定病原为坦布苏病毒(Tembusu virus,TMUV)。坦布苏病毒主要感染鸭,但也能感染鹅,造成鹅的发病和死亡。种鹅感染TMUV后严重影响种鹅产蛋量,雏鹅感染坦布苏病毒发病严重,生长迟缓,最终可导致部分发病严重雏鹅死亡,已经给我国养鹅业带来巨大的经济损失。本研究用实验室分离到的鸭源坦布苏病毒分别对5日龄和20日龄的雏鹅进行人工感染试验,研究该病毒对雏鹅的致病性,旨在为今后鹅坦布苏病毒感染的防控提供理论依据。100只1日龄健康雏鹅平均分为5组,每组20只。1-4组为实验组,5组为对照组。1-2组于5日龄、3-4组于20日龄分别经静脉和点眼滴鼻人工接种TMUV SDSG株(ELD50为10-2.17/0.2mL),5组接种等剂量的生理盐水。观察鹅的发病情况和临床症状,在攻毒后第3d、6d、9d、12d于各组随机选取3只雏鹅,采血并剖杀,观察剖检变化,采集脑、肝脏、肺脏、胰脏、脾脏等,进行病理组织学观察、载毒量分析和血液细胞因子测定。结果显示:发病鹅临床症状主要表现为,5日龄静脉注射攻毒组自攻毒后第4d开始发病,点眼滴鼻组自攻毒后第5d开始发病,发病鹅出现精神沉郁,厌食,排白绿色、棕色稀便,两腿无力,站立不稳,颤抖,生长迟缓,伴有腹部朝上两腿挣扎呈游泳状,角弓反张直至死亡等症状,偶见静脉注射组发病鹅出现头颈震颤神经症状。自攻毒后第5d静脉注射组有病鹅出现死亡,攻毒后第6d点眼滴鼻组开始出现死亡,发病时间集中在攻毒后4-8d,死亡时间集中在攻毒后的5-8d,从第9d开始临床症状逐渐减轻。20日龄攻毒组自攻毒5d后开始出现食欲下降,精神不振,排黄绿色稀便,病程维持3d左右,攻毒后8-9d临床症状消失,采食量回升。分别在攻毒后第3d、6d、9d、12d测定攻毒鹅和对照鹅的体重,5日龄攻毒组较20日龄攻毒组发病鹅体重下降更为明显。病死鹅剖检病变为多个组织器官的出血和坏死,包括脑膜充血、出血、脑水肿;心冠脂肪出血、心内膜出血、心肌坏死;肺水肿、出血、淤血;腺胃乳头轻度出血;肝脏出血、坏死;胰腺水肿、坏死;脾脏肿大、出血;肾脏出血、水肿等病变。病理组织学变化主要包括,脑血管周围间隙变大水肿、脑膜有大量炎性细胞浸润、充血、脑间质散在小胶质细胞增生;肝、胰腺、肾脏的腺管上皮细胞凋亡、坏死,腺管间隙扩张淤血;脾脏大量淋巴细胞凋亡空泡化;心肌断裂、变性、坏死、心肌纤维间距变大并伴有炎性细胞浸润;肺出血、淤血等病变明显。实时荧光定量PCR检测结果显示,胰腺和脑是病毒载量最高且持续时间最长的器官,心、脾、肾、肠、胸腺、法氏囊、腺胃含量则次之,相对表达量较低的器官主要是肝和肺。攻毒3d后各组织均可检测到病毒,攻毒6 d后病毒载量达到高峰,随后病毒含量逐渐降低,攻毒后第12d肝、肺、胸腺和腺胃检测不到病毒。比较5日龄攻毒组和20日龄攻毒组各组织器官的病毒载量,5日龄攻毒组是20日龄攻毒组的10倍以上。细胞因子测定结果显示:测定IL-4,IFN-γ结果显示,细胞因子水平呈现先下降后上升的总趋势。人工感染雏鹅第3d,IL-4、IFN-γ的含量与对照组相比无明显差异性;第6-9d,IL-4、IFN-γ含量低于对照组,自第9d开始IL-4、IFN-γ的含量开始逐渐上升,第12d可见IL-4、IFN-γ的含量高于对照组。抗体水平测定结果显示:在攻毒后第3d未检测到坦布苏病毒抗体,第6d可检测到抗体,随后抗体水平呈上升趋势直至第12d依然上升。综上所述,坦布苏病毒对雏鹅致病性的显著,5日龄试验组临床症状、剖检病变和病理组织学变化明显显著于20日龄试验组。因此,在生产中,应做好鹅坦布苏病毒感染的防控。更多还原

【Abstract】 In China many egg-laying ducks farms and breeder geese farms had suffered from a serious infectious disease with the main clinical symptoms in sever egg production decrease since April 2010, and had identified the pathogen was TMUV(Tembusu Virus). TMUV infection ducks primarily, while it’s could infection in geese and caused infected geese morbidity and death. Breeder geese infected in TMUV mainly lead to egg production decrease obviously, while the results of goslings inoculated in TMUV is with highly morbidity, many serious pathogenetic goslings went to death ultimately, and had caused to a huge economic loss in our countries goose industry. To investigate the pathology of TMUV in goslings, this study was chose TMUV-SDSG strain inoculated in 5-day-old goslings and 20-day-old goslings respectively, and TMUV-SDSG Strain was isolated in ducks from the institute of poultry disease at Shandong Agricultural University. The purpose of this study is to provide a theoretical basis to prevent and control TMUV for goslings in future.Total 100 healthy 1-day-old goslings were equally divided in 5 groups, group one to group four are experimental groups, group five is the control goslings. Group one and two are5-day-old goslings, group three and four are 20-day-old goslings were inoculated with TMUV-SDSG strain(ELD50 10-2.17/0.2m L) in intravenous injection and intranasal drip,respectively. Group 5 goslings were inoculated with the same volume of sterile physiological saline. After infected in TMUV autopsies observed the autopsy symptoms congestion, each group goslings were chose 3 randomly and made periodically to get brains, livers, lungs,pancreas, spleens and other tissues from to at 3dpi, 6dpi, 9dpi, and 12 dpi. Then from the follow three aspects such as to observe the histopathological lesions, the viral load through real time RT-PCR and cytokines test to investigate the pathogenicity of Tembusu Virus for goslings.The results showed that the primarily clinical signs of 5-day-old goslings infected in TMUV through intravenous injection attacked at 4dpi, and the young gosling inoculated TMUV in intranasal drip attacked at 5 dpi. All morbidity goslings were with the main clinical signs including listlessness, inappetence, passage white and green loose stools, legs-weak,marked incoordination, tremble, slow-growing, belly upward with legs struggle as swimming,opisthotonos, and then to death, while the signs of head and neck tremble was showed occasionally. Goslings infected in TMUV through intravenous injection began to death at 5dpi and the intranasal drip groups began to death at 6di. The morbidity and mortality time were focused on 4~8 dpi and 5~8dpi, respectively. And the clinical signs began to relieve at 9dpi.20-old-day goslings infected in TMUV began to showed clinical signs at 5dpi with through intravenous injection, and the course of disease last about 3dpi, however the clinical signs disappear at 8~9 dpi in feed intake rise again. To compare the body weight between 5-day-old goslings and 20-day-old goslings infected in TMUV showed that the younger groups loose higher weight than the older goslings relative to the control goslings at 3dpi, 6dpi, 9dpi,and12 dpi.During autopsy symptoms the gross lesions mainly included congestion, hemorrhagic and dropsical. Gross lesions were observed in most infected goslings organs included hyperaemia,meningorrhagia and encephalitis in brain, myocardial intima haemorrhages and necrosis,hyperaemia in glandular stomach nipple, pneumonedema and pulmonary congestion in lungs,hyperaemia and necrosis in livers, necrosis foci in pancreas, splenomegaly and haemorrhages in spleen, haemorrhages in kidney and so on.The primary microscopic lesions were congestion, hemorrhage, necrosis, massive infiltration of mononuclear cells consisting of lymphocytes, plasma cells, and histiocytes and lymphoid nodules. The lesions of brain showed a viral encephalitis change with perivascular space increased, encephaledema, glialnodule and massive microglia proliferation in brain parenchyma. The congestion of vessel, massive infiltration of mononuclear cells and massive inflammatory cells infiltration were found in the cerebral cortex at. The lesions of liver,kidney, pancreas mainly consisted of degenerate tubules with vacuolated epithelial cells,hyperemia and haemorrhage, and inflammatory cell disintegration. Spleen lesions contained hemorrhage, massive lymphocytes. Lesions of heart included cardiac muscle cells disruption,cardiac muscle fibers hemorrhage, massive infiltration by mononuclear cells, fragmetation of the myocardium and myocyte necrosis. And finally the lesions of lung included haemorrhage and pulmonary congestion.According to the above, qRT-PCR were detected to investigated the antigen of TMUV distributions in different tissues at 3dpi, 6dpi, 9dpi, and 12 dpi. The result showed that and the highest level of viral copies was found in brain and pancreas, heart, spleen, kidney, intestines,thymus, bursa and glandular stomach were take second place,while the lowest were liver and lung. All tissues could detect the relative expression of TMUV at 3 dpi, and the viral load achieved peck at 6dpi, and then to decrease. The relative expression didn’t detect at 12 dpi in liver, lung, thymus and glandular stomach. To compared 5-day-old infected groups and20-day-old groups, the viral load in the younger goslings are more than 10 times as the20-day-old goslings.The result of IL-4 and IFN-γ showed that, the load of IL-4 and IFN-γgoslings infected in TMUV was lower than the control group at 3 dpi and 6dpi, and then began to raised up at9 dpi. The infected goslings didn’t test obvious differences compared with the healthy geese at3 dpi, and the load of IL-4 and IFN-γ began to higher than the control group at 12 dip.The result of antibody levels showed that the serum could detected TMUV antibody levels at 6dpi, then reached at 9dpi and 12 dpi.In conclusion, goslings were sensitive to TMUV with that not only did age-related differences in the resistance to TMUV infection were observed younger geese being more susceptible, but also infected ways diversity could influenced the outcome that intravenous injection geese were more severe than intranasal drip geese. So in clinical process, we should pay more attention to the pathogenicity of TMUV for goslings, and do well operation for TMUV prevention and treatment.更多还原