番鸭呼肠孤病毒诱导的细胞凋亡观察

以番鸭呼肠孤病毒人工感染10日龄健康番鸭,运用原位末端标记技术及免疫组织化学方法,对番鸭呼肠孤病毒感染后番鸭多种组织的细胞凋亡进行检测,对死亡因子FasL的表达进行研究。原位末端标记检测显示,多种组织器官中出现大量阳性细胞,表明番鸭呼肠孤感染可引起肝脏、脾脏、肾脏、肺脏、胸腺、盲肠和法氏囊发生不同程度的细胞凋亡。免疫组织化学研究结果表明,感染番鸭呼肠孤病毒后,肝脏、肾脏、肺脏、胸腺、盲肠和法氏囊等多种组织中可观察到FasL表达,且各种组织FasL表达的强弱与原位末端标记检测的细胞凋亡指数高低相一致。结果表明番鸭呼肠孤病毒诱导细胞凋亡的机制与FasL的表达密切相关。     

番鸭呼肠孤病毒诱导雏番鸭免疫器官细胞凋亡的研究

以番鸭呼肠孤病毒腿部肌肉接种10龄健康雏番鸭,复制番鸭"花肝病",并研究其不同时期诱导免疫器官细胞凋亡的能力.结果表明,雏番鸭在感染病毒后的12 h、24 h、48 h、72 h、144 h均能观察到胸腺、脾脏、法氏囊细胞凋亡的典型形态学变化,在12～24 h达到高峰,72 h后逐渐降低.由此可知,雏番鸭呼肠孤病毒对雏鸭免疫器官的细胞凋亡具有诱导作用.揭示该病毒致病机理与淋巴细胞凋亡从而引起的免疫抑制相关.     

番鸭呼肠孤病毒感染雏番鸭的病理学观察

对呼肠孤病毒感染雏番鸭进行了肉眼病变观察和组织病理切片检查。结果显示,雏番鸭感染番鸭呼肠孤病毒后,除机体出现临床症状外,主要呈现明显的脚软、腹泻、生长障碍为主要症状;病理剖检发现肝、脾表面坏死、纤维素性心包炎为主要病变;组织病理学检查发现肝脏汇管区出现明显卵圆细胞及胆管细胞增生,汇管区细胞肿胀变性、呈空泡状;脾脏脾小体中心溶解坏死;心脏纤维素性心外膜炎;肾脏异嗜性白细胞浸润;其他器官无明显的异常与病理变化完全相符。     

禽流感病毒与番鸭呼肠病毒感染番鸭免疫器官细胞凋亡的研究

应用原位末端标记法和免疫组化SABC法,分别以H9亚型禽流感和番鸭呼肠病毒单独感染或混合感染雏番鸭后1、3、5、7、10d对胸腺、法氏囊、脾脏细胞凋亡和P53的动态变化进行检测。结果显示,H9亚型AIV,MDRV单独感染和混合感染均可导致雏番鸭胸腺、法氏囊和脾脏出现淋巴细胞凋亡增多;病毒感染早期各免疫器官组织细胞凋亡明显,感染后期细胞凋亡量逐渐减少;混合感染组脾脏、胸腺细胞凋亡更为显著。P53表达的变化与细胞凋亡呈现相同变化规律,表明P53表达与病毒诱导的细胞凋亡机制密切相关。     

番鸭呼肠孤病毒强弱毒株对番鸭免疫器官形态学的影响

本文探讨番鸭呼肠孤病毒强弱毒株对番鸭免疫器官超微结构的影响。强毒株感染番鸭显微结构变化表现为:各器官不同程度变性、细胞溶解坏死及血管扩张充血,病灶区及血管周围淋巴单核细胞明显浸润;免疫器官脾、胸腺和法氏囊淋巴细胞变性坏死,数量明显减少。脾脏淋巴细胞坏死,数量减少,网状结缔组织显露。而弱毒疫苗株接种后番鸭胸腺、法氏囊、脾脏组织学形态与健康对照鸭一致,均未发现在显微结构上的损伤。表明番鸭呼肠孤病毒强毒严重损伤免疫器官,而弱毒不仅失去对雏番鸭的致病性,而且也失去了损伤免疫器官和免疫抑制能力。     

番鸭呼肠孤病毒病雏番鸭实质器官的超微结构

对人工感染番鸭呼肠孤病毒发病雏番鸭的心、肝、肺、肾、脾脏、胸腺、法氏囊等7种实质器官的超微结构进行了观察。电镜下发现：心、肝、肺、肾等实质器官出现不同程度的细胞变性、水肿以及局灶性溶解坏死；各器官血管内皮细胞脂滴增多、水肿以至坏死脱落．通透性增加；浆细胞、淋巴细胞和吞噬细胞呈散在或灶性浸润于坏死区和实质细胞间。免疫器官脾脏、胸腺和法氏囊中的部分淋巴细胞、浆细胞坏死和不同程度凋亡，且细胞溶解坏死形成大小不一的坏死灶并被大量增生的吞噬细胞所吞噬，淋巴细胞数量明显减少。上述结果提示，番鸭呼肠孤病毒能导致番鸭免疫抑制。     

一例雏番鸭新型鸭呼肠孤病毒感染的诊断

2017年1月,福建省南平市延平区某养殖户饲养的雏番鸭出现大面积死亡,死亡率约45%。死亡鸭见有角弓反张、运动失调等症状。剖检死亡雏番鸭见肝脏呈现大面积点状、片状不规则出血,且伴有坏死;脾脏见有明显的脾坏死。结合临床初步诊断为新型鸭呼肠孤病毒感染。采集病死雏番鸭的肝脏和脾脏,进行细菌分离鉴定与新型鸭呼肠孤病毒RT-PCR检测。结果表明,死亡雏番鸭细菌分离为阴性,RT-PCR试验可以特异性扩增出约586 bp的新型鸭呼肠孤病毒特异性条带,而经典呼肠孤病毒和鸭肝炎病毒未见特异性目的条带,确诊雏番鸭感染新型鸭呼肠孤病毒。     

番鸭呼肠孤病毒病活疫苗诱导的免疫应答分析

本研究旨在评价番鸭呼肠孤病毒病活疫苗对番鸭免疫功能的影响,探讨番鸭呼肠孤病毒病活疫苗的免疫机理;1日龄番鸭免疫番鸭呼肠孤病毒病活疫苗,分别于免疫后7～35 d观察番鸭生长发育,检测免疫番鸭血清中和抗体效价,外周血淋巴细胞、胸腺细胞和法氏囊细胞对ConA、LPS的反应,细胞毒T细胞(CTL)的细胞毒性作用.结果显示:免疫番鸭生长发育正常,免疫器官中胸腺明显增大;免疫后35 d血清中和抗体才开始上升;外周血和胸腺淋巴细胞对ConA的增殖反应显著提高(P＜0.05);而外周血和法氏囊淋巴细胞对LPS增殖反应无显著变化(P＜0.05);外周血细胞毒性T细胞杀伤功能增强(P＜0.05).结果提示番鸭呼肠孤病毒病活疫苗免疫后主要刺激提高番鸭细胞免疫功能,提高机体抵抗番鸭呼肠孤病毒的能力.     

不同感染剂量MDRV对番鸭免疫反应和细胞毒性作用的影响

用不同剂量番鸭呼肠孤病毒(MDRV MW9710株)感染8日龄番鸭后,通过检测血液中淋巴细胞对ConA、LPS的反应和NK细胞、细胞毒T细胞(CTL)的细胞毒性作用,探讨MDRV感染对番鸭免疫细胞功能和细胞毒性作用的影响。结果显示,不同感染剂量MDRV均会抑制番鸭血液淋巴细胞对ConA、LPS的增殖反应,降低NK细胞和CTL细胞的杀伤活性,且影响程度与剂量相关;同时感染番鸭生长缓慢,脾脏肿大,胸腺和法氏囊缩小。上述结果表明,MDRV感染能导致番鸭免疫抑制,且细胞免疫抑制程度与感染病毒量有关。     

MTS法检测番鸭呼肠孤病毒对鸭胚成纤维细胞活力的影响

采用MTS法检测接种不同致病力番鸭呼肠孤病毒12h、24h、36h、48h、72h、96h后对番鸭鸭胚成纤维细胞(MDEF)活力的影响。根据细胞病变情况和活力状态确定细胞发生变化的拐点,初步探讨番鸭呼肠孤病毒感染诱导细胞凋亡的发生和细胞活力的特征变化,进一步了解番鸭呼肠孤病毒与宿主之间的相互关系,为研究番鸭呼肠孤病毒感染和致病机理提供时间科学依据。结果表明,接毒后36h,细胞活力达到最高,高致病力YB毒株作用于MDEF,细胞活力在48h大幅下降并出现明显细胞凋亡;低致病力YJL毒株作用于MDEF,则在72h可出现明显细胞凋亡。     

Comparative pathological studies on domestic geese (Anser anser domestica) and Muscovy ducks (Cairina moschata) experimentally infected with parvovirus strains of goose and Muscovy duck origin

Parvovirus infection of Muscovy ducks caused by a genetically and antigenically distinct virus has been reported from Germany, France, Israel, Hungary, some Asian countries and the USA. The pathological changes include those of degenerative skeletal muscle myopathy and myocarditis, hepatitis, sciatic neuritis and polioencephalomyelitis. In the study presented here, day-old and 3-week-old goslings and Muscovy ducks were infected experimentally with three different parvovirus strains (isolates of D-216/4 from the classical form of Derzsy's disease, D-190/3 from the enteric form of Derzsy's disease, and strain FM from the parvovirus disease of Muscovy ducks). All three parvovirus strains caused severe disease in both day-old and 3-week-old Muscovy ducks but in the goslings only the two strains of goose origin (D-216/4 and D-190/3) caused disease with high (90-100%) mortality when infection was performed at day old. Strain FM (of Muscovy duck origin) did not cause any clinical signs or pathological lesions in the goslings. In the day-old goslings and Muscovy ducks the principal pathological lesions were severe enteritis with necrosis of the epithelial cells (enterocytes) of the mucous membrane and the crypts of Lieberkühn, and the formation of intranuclear inclusion bodies. Other prominent lesions included hepatitis and atrophy (lymphocyte depletion) of the lymphoid organs (bursa of Fabricius, thymus, spleen). In goslings infected with the strain originating from the classical form of Derzsy's disease mild myocarditis was also detected. After infection at three weeks of age, growth retardation, feathering disorders, myocardial lesions (degeneration of cardiac muscle cells, lympho-histiocytic infiltration) and hepatitis were the most prominent lesions in both geese and Muscovy ducks. In addition to the lesions observed in the geese, muscle fibre degeneration, mild sciatic neuritis and polioencephalomyelitis were also observed in the Muscovy ducks infected with any of the three parvovirus strains.     

Muscovy duck reovirus infection rapidly activates host innate immune signaling and induces an effective antiviral immune response involving critical interferons

Muscovy duck reovirus (MDRV) is a highly pathogenic virus in waterfowl and causes significant economic loss in the poultry industry worldwide. Because the host innate immunity plays a key role in defending against virus invasion, more and more attentions have been paid to the immune response triggered by viral infection. Here we found that the genomic RNA of MDRV was able to rapidly induce the production of interferons (IFNs) in host. Mechanistically, MDRV infection induced robust expression of IFNs in host mainly through RIG-I, MDA5 and TLR3-dependent signaling pathways. In addition, we observed that silencing VISA expression in 293T cells could significantly inhibit the secretion of IFNs. Remarkably, the production of IFNs was reduced by inhibiting the activation of NF-κB or knocking down the expression of IRF-7. Furthermore, our study showed that treatment of 293T cells and Muscovy duck embryo fibroblasts with IFNs markedly impaired MDRV replication, suggesting that these IFNs play an important role in antiviral response during the MDRV infection. Importantly, we also detected the induced expression of RIG-I, MDA5, TLR3 and type I IFN in Muscovy ducks infected with MDRV at different time points post infection. The results from in vivo studies were consistent with those in 293T cells infected with MDRV. Taken together, our findings reveal that the host can resist MDRV invasion by activating innate immune response involving RIG-I, MDA5 and TLR3-dependent signaling pathways that govern IFN production.     

CFSE标记法分析番鸭呼肠孤病毒感染对番鸭回肠淋巴细胞归巢的影响

旨在建立一种可靠的体外活番鸭淋巴细胞的荧光标记方法,并用于分析番鸭呼肠孤病毒感染对雏番鸭回肠淋巴细胞归巢的影响。选择活细胞荧光染剂5（6）-羧基二乙酸荧光素琥珀酰亚胺酯（CFDA-SE,CFSE）对分离的番鸭淋巴细胞进行标记和利用流式细胞术对体外标记的淋巴细胞体内示踪检测分析,并利用该方法对MDRV感染雏番鸭回肠组织的淋巴细胞数量进行流式细胞术和石蜡切片免疫荧光检测分析;结果最终确定CFSE体外标记番鸭淋巴细胞的条件为以PBS为孵育液,终浓度10 μmol·L^-1,37℃ 30 min;试验结果表明番鸭外周血内的CFSE⁺淋巴细胞率基本稳定在2%~5%,CFSE⁺淋巴细胞峰值出现顺序依次为脾、空肠、回肠、盲肠、十二指肠、法氏囊和胸腺;此外,感染MDRV后1~10 d MDRV组中CFSE⁺淋巴细胞率极显著（P<0.01）高于MOCK组,该结果与α4⁺淋巴细胞率和石蜡切片免疫荧光检测结果一致。结果表明本试验CFSE标记的淋巴细胞可用于体内淋巴细胞示踪,初步应用结果提示MDRV感染促进番鸭淋巴细胞向回肠归巢,为进一步阐明MDRV感染的肠道组织致病机制奠定了基础。     

番鸭感染“白点病”病毒后血浆总胆红素和某些酶的动态变化

以番鸭“白点病”病毒株人工感染雏番鸭 ,于感染后 1 2 ,2 4 ,48,1 2 0 ,1 68,2 4 0和 360h分别检测对照组和感染组番鸭的血浆总胆红素(TBil)含量和碱性磷酸酶 (AKP) ,丙氨酸氨基转移酶 (ALT)和天冬氨酸氨基转移酶 (AST)的活性 ,同时观察番鸭的临床症状和病理变化。结果 :感染组番鸭的Tbil含量在感染后 1 2和 2 4 0h显著低于对照组 (P<0 0 5) ;AKP活性在攻毒后 1 2h开始显著上升 ,并持续至试验结束 ;ALT活性在攻毒后 2 4 ,1 2 0和 1 68h显著上升 ;AST活性在 1 2 ,48,1 2 0和 2 4 0h显著升高。番鸭感染“白点病”后血浆中酶活性上升 ,提示肝脏、心脏等器官受损明显。感染后期血浆ALT和AST活性趋于正常 ,表明受损器官在后期逐渐恢复。这与临床症状和病理变化相对应。     

不同毒力番鸭呼肠孤病毒在番鸭免疫器官的分布和排毒

探讨番鸭呼肠孤病毒强毒株和弱毒株在番鸭免疫器官中的分布和排毒的差异。结果显示强毒株在感染后1d就可在脾脏、法氏囊、胸腺检出病毒RNA,高峰期为攻毒后7～14d;直到感染后35d,在免疫器官中不能检测到病毒RNA。接种强毒株后7d开始向外界排毒,而14d后停止向外界排毒。雏鸭免疫弱毒疫苗后3d,即可在脾、胸腺、法氏囊中检出病毒RNA;高峰期为攻毒后7～14d,免疫后21d免疫器官中的检测逐渐降低,直到感染后28d,在免疫器官中不能检测到病毒RNA。表明番鸭接种活疫苗后7d开始向外界排毒,而11d后停止向外界排毒。     

番鸭细小病毒病病原的血清学检测与PCR鉴定

为了解安徽省番鸭细小病毒病的流行情况,采用酶联免疫吸附试验(ELISA)对该省内部分番鸭群进行鹅细小病毒的血清抗体检测,同时对疑似病鸭进行了病原的PCR检测。结果发现,在被检测的44份血清样本中鹅细小病毒血清抗体阳性率高达34.1%;PCR检测结果显示,所检测的样品中,有2份为鹅细小病毒阳性,1份为番鸭细小病毒阳性,且3份被检样品均来自雏番鸭。该研究结果表明,该省番鸭群中存在鹅细小病毒和番鸭细小病毒感染,应采取有效的预防控制措施。     

Isolation and characterization of a subtype C avian metapneumovirus circulating in Muscovy ducks in China

Subtype C avian metapneumovirus (aMPV-C), is an important pathogen that can cause egg-drop and acute respiratory diseases in poultry. To date, aMPV-C infection has not been documented in Muscovy ducks in China. Here, we isolated and characterized an aMPV-C, designated S-01, which has caused severe respiratory disease and noticeable egg drop in Muscovy duck flocks in south China since 2010. Electron microscopy showed that the isolate was an enveloped virus exhibiting multiple morphologies with a diameter of 20–500 nm. The S-01 strain was able to produce a typical cytopathic effect (CPE) on Vero cells and cause death in 10- to 11-day-old Muscovy duck embryos. In vivo infection of layer Muscovy ducks with the isolate resulted in typical clinical signs and pathological lesions similar to those seen in the original infected cases. We report the first complete genomic sequence of aMPV-C from Muscovy ducks. A phylogenetic analysis strongly suggested that the S-01 virus belongs to the aMPV-C family, sharing 92.3%-94.3% of nucleotide identity with that of aMPV-C, and was most closely related to the aMPV-C strains isolated from Muscovy ducks in France. The deduced eight main proteins (N, P, M, F, M2, SH, G and L) of the novel isolate shared higher identity with hMPV than with other aMPV (subtypes A, B and D). S-01 could bind a monoclonal antibody against the F protein of hMPV. Together, our results indicate that subtype-C aMPV has been circulating in Muscovy duck flocks in South China, and it is urgent for companies to develop new vaccines to control the spread of the virus in China.     

雏番鸭细小病毒病显微和超微结构研究

应用光镜和电镜技术研究了人工感染雏番鸭细小病毒病死鸭的心、肝、脾、肺、肾、胰、法氏囊和大脑等8种器官的显微结构和发病鸭的心、肝、脾、肺、肾、胰等6种器官的超微结构变化。结果如下：1．显微结构变化主要表现为：各器官的血管扩张、充血，以肺脏尤为严重，并见少量淋巴单核细胞浸润，同时心、肝、肾、胰和大脑均呈现不同程度的变性；而免疫器官脾、法氏囊则主要表现为淋巴细胞数量减少。2．超微结构变化主要表现为：（1）各实质细胞内线粒体肿胀，嵴变少而或崩解，内质网减少，脂滴增多，部分细胞水肿坏死；（2）血管内皮均有不同程度的脂滴增多，水肿以至脱落；（3）吞噬能力加强，表现为细胞内次级溶酶体增多，吞噬细胞数量增加，其胞浆内含大量的吞噬体。