一株鸡包涵体肝炎病毒的分离鉴定

利用鸡包涵体肝炎自然病例的肝组织，通过ＳＰＦ鸡胚传代和鸡胚肾遥代细胞培养，分离到一株鸡包涵体肝炎病毒Ｈｂ株。该病毒株的复制可被５－溴尿嘧啶－２‘脱氧核苷抑制，对乙醚和氯仿有低抗力，说明Ｈｂ株的核酸型为ＤＮＡ，无脂质囊膜。     

鸡包涵体肝炎鸡胚肝细胞包涵体特性的研究

应用鸡包涵体肝炎病毒 FAV- Hb株试验感染 SPF鸡胚 ,通过透射电镜对感染鸡胚肝脏的观察 ,表明 FAV- Hb株可引起鸡胚肝细胞内形成三种类型的包涵体 ,即非病毒性中等电子密度包涵体 ,病毒性包涵体和非病毒性高电子密度包涵体 ,各型包涵体均可见于胞核或胞浆内 ,但胞核是包涵体首先形成的部位 ,核内包涵体通过核膜进入胞浆。各型包涵体在其形成过程中有较密切的关系。     

鸡包涵体肝炎病原检测及病理学观察

取临床疑似鸡包涵体肝炎病例的肝组织,接种SPF鸡胚获取尿囊液,对获取的尿囊液进行PCR扩增和基因测序分析,成功分离并鉴定出1株鸡包涵体肝炎病毒,同时对送检病例采样进行病理组织学观察。将病死鸡肝组织研磨后,经尿囊腔接种9日龄的SPF鸡胚,用获得的尿囊液提取病毒DNA,成功获得DNA模板。根据已发表的Ⅰ群禽腺病毒hexon全基因的保守区域设计并合成1对引物,用这对引物对病毒的DNA模板进行PCR扩增,结果扩增出与目的基因大小一致的片段。病理学观察见肝组织淤血,细胞核浓缩,发生坏死,核内出现包涵体,伴有脂肪变性。测序分析表明,分离株与Ⅰ群禽腺病毒中血清4型相似性为98.6%,与其他血清型的相似性为68.7%~96.9%,与Ⅱ群禽腺病毒的鸡出血性肠炎病毒和Ⅲ群禽腺病毒的减蛋综合征病毒的相似性为44.6%,最终确定分离病毒为Ⅰ群禽腺病毒,为进一步鉴定和分离Ⅰ群禽腺病毒提供了依据。     

一株鸡包涵体肝炎病毒的分离鉴定

利用鸡包涵体肝炎自然病例的肝组织，通过ＳＰＦ鸡胚传代和鸡胚肾原代细胞培养，分离到一株鸡包涵体肝炎病毒Ｈｂ株。该病毒株的复制可被５－溴尿嘧啶－２′脱氧核苷抑制，对乙醚和氯仿有低抗力，说明Ｈｂ株的核酸型为ＤＮＡ，无脂质囊膜。Ｈｂ株对酸有抵抗力，对热敏感。电镜观察表明，病毒粒子呈球形，直径约８０～９０ｎｍ。用ＡＡＶ－２、３、４、５、８型的分型高免血清进行中和试验，证明Ｈｂ株为血清型８型。回归试验表明Ｈｂ株具有较强的致病性     

浅谈禽腺病毒感染鸡的特征及诊断

1949年，Van den Ende分离出一株禽腺病毒。以后．又从鸡胚中无意分离到多株鸡胚致死孤儿(Chicken embayo lethal orphan CELO)病毒。1952年Olson自患呼吸道疾病(支气管炎)的北美鹌鹑病料中分离获得了第一株发病禽的腺病毒，即鹌鹑支气管炎病毒(QBV)。鸡类腺病毒(A viadeno vires)包括鸡胚致死孤儿病毒(CELOV)，包涵体肝炎病毒(IBHV)，减蛋综合症(EDS-76)。     

一株高致病性血清4型禽腺病毒的分离与鉴定

研究对临床上疑似禽腺病毒感染鸡病例进行了病毒分离与鉴定。通过对病鸡肝组织研磨液接种鸡胚、电镜观察、PCR及序列鉴定,分离出一株血清4型禽腺病毒,命名为JH。动物回归试验证明,肌肉接种1 000 EID_(50)病毒量对3周龄SPF鸡的致死率高达90%;病死鸡剖解可见典型的心包积液与包涵体肝炎,肝组织切片HE染色可见典型的嗜碱性核内包涵体。这一高致病性血清4型禽腺病毒的分离报道,为探究国内禽腺病毒分子流行与毒力演变提供基础材料。     

2株Ⅰ群禽腺病毒的分离鉴定及致病性分析

为了解山东省禽腺病毒(fowl adenovirus,FAdV)毒株的基因遗传演化情况及致病性,本试验对山东省两家疑似暴发鸡包涵体肝炎和心包积液综合征鸡场采集的病料(肝脏、脾脏)进行PCR鉴定,并将鉴定为FAdV的2份阳性病料提取病毒液,接种鸡肝癌细胞(LMH)进行毒株传代培养和细胞病变(CPE)观察、PCR检测、TCID50测定、鸡胚致病性试验、SPF鸡回归试验、病毒hexon部分基因扩增及序列分析。结果显示,试验成功分离到2株FAdV,2株分离株细胞传第1代即可观察到CPE,PCR均可扩增出大小为500bp的片段,且2株分离株细胞F5代TCID50分别为10-7.75/0.1mL和10-7.60/0.1mL,均对7日龄SPF鸡胚有强致病性;第1分离株和第2分离株对21日龄SPF鸡攻毒死亡率分别为80%和15%。hexon基因核苷酸同源性分析结果显示,第1分离株与FAdV-4株同源性最高(99.57%),第2分离株与FAdV-8b株同源性最高(99.18%)。这2株FAdV分离株均与Ⅰ群禽腺病毒同源,与火鸡出血性肠炎病毒(HEV)所在的血清Ⅱ群及减蛋综合征病毒(EDSV)所在的血清Ⅲ群禽腺病毒位于不同分支。第1分离株基因型为C型,血清型为4型,命名为FAdV-SDC4株;第2分离株基因型为E型,血清型为8b型,命名为FAdV-SDE8b株。综上所述,FAdV-SDC4和FAdV-SDE8b株属于近几年国内流行毒株。本研究结果可为鸡包涵体肝炎、心包积液综合征的防控工作和疫苗株的选择提供科学依据。     

鸡包涵体肝炎病毒CELOV 株的鉴定研究

采用鸡胚有限稀释法将鸡包涵体肝炎病毒（CELOV）纯化。将纯化的CELOV在SPF鸡胚上连续传10代,对各代次的毒种均进行毒价测定,并选择不同代次进行外源病毒、抗原性等方面的系统鉴定。结果表明,不同代次的病毒毒价稳定,病毒纯净、特异,无外源病毒感染。采用不同代次病毒鸡胚尿囊液制备3批琼脂扩散试验抗原敏感、特异,用原倍、2倍稀释的抗原可以与32倍稀释的阳性血清反应,出现清晰的沉淀线。抗原不与其它病原体的阳性血清反应。用CELOV制备的琼扩抗原可用于鸡包涵体肝炎病毒感染的血清学检测。     

鸡痘病毒的分离鉴定

利用鸡胚毛尿囊膜培养、透射电镜观察、包涵体检查及抗原检测，从某种鸡场发病鸡群的鸡冠部痘疹上分离鉴定出两株鸡痘病毒。两株病毒能在尿囊膜上形成痘斑和包涵体，病毒粒子大小为２５０×３５０ｎｍ，具有囊膜和血凝，对乙酰，氯仿和ＰＨ３敏感，５６℃３０ｍｉｎ可灭活，人工感染试验结果表明，两株病毒具有较强的致病性。     

2株Ⅰ群禽腺病毒的分离鉴定及致病性分析

为了解山东省禽腺病毒（fowl adenovirus,FAdV）毒株的基因遗传演化情况及致病性,本试验对山东省两家疑似暴发鸡包涵体肝炎和心包积液综合征鸡场采集的病料（肝脏、脾脏）进行PCR鉴定,并将鉴定为FAdV的2份阳性病料提取病毒液,接种鸡肝癌细胞（LMH）进行毒株传代培养和细胞病变（CPE）观察、PCR检测、TCID₅₀测定、鸡胚致病性试验、SPF鸡回归试验、病毒hexon部分基因扩增及序列分析。结果显示,试验成功分离到2株FAdV,2株分离株细胞传第1代即可观察到CPE,PCR均可扩增出大小为500 bp的片段,且2株分离株细胞F5代TCID₅₀分别为10^-7.75/0.1 mL和10^-7.60/0.1 mL,均对7日龄SPF鸡胚有强致病性;第1分离株和第2分离株对21日龄SPF鸡攻毒死亡率分别为80%和15%。hexon基因核苷酸同源性分析结果显示,第1分离株与FAdV-4株同源性最高（99.57%）,第2分离株与FAdV-8b株同源性最高（99.18%）。这2株FAdV分离株均与Ⅰ群禽腺病毒同源,与火鸡出血性肠炎病毒（HEV）所在的血清Ⅱ群及减蛋综合征病毒（EDSV）所在的血清Ⅲ群禽腺病毒位于不同分支。第1分离株基因型为C型,血清型为4型,命名为FAdV-SDC4株;第2分离株基因型为E型,血清型为8b型,命名为FAdV-SDE8b株。综上所述,FAdV-SDC4和FAdV-SDE8b株属于近几年国内流行毒株。本研究结果可为鸡包涵体肝炎、心包积液综合征的防控工作和疫苗株的选择提供科学依据。     

鸡包涵体肝炎肿瘤坏死因子消长规律的研究

自1975年从动物体内提取肿瘤坏死因子（TNF）以来，许多科学家从哺乳动物及禽类体内提取TNF并对其理化性质、生物学活性进行了不同程度的研究和探讨。而用鸡包涵体肝炎病毒诱生TNF与该病之间相关性及产生的消长规律的研究尚属空白。本研究应用鸡包涵体肝炎病毒（FAV-HA毒株）接种SPF雏鸡，在接种后的不同日龄采血，提取TNF，并对其免疫活性（对L₉₂₉细胞的杀伤率）进行了检测，从而进一步研究其产生TNF的消长规律及与该病毒的相关性。研究结果表明，鸡包涵体肝炎病毒经口腔感染SPF雏鸡可刺激机体产生TNF，在感染的不同日龄鸡的TNF活性出现一定的消长规律。这进一步说明鸡包涵体肝炎(IBH)与TNF的产生及其活性具有密切的相关性。     

Characterisation of avian adenoviruses associated with inclusion body hepatitis

Eleven avian adenoviruses were isolated in monolayer cultures of specific pathogen free chicken kidney cells which were inoculated with suspensions of liver, intestine or bursa obtained from 15 broiler flocks experiencing outbreaks of inclusion body hepatitis (10 isolates) and from five unaffected flocks (one isolate). Of the 11 isolates obtained, nine were identified by virus neutralisation tests as serotype 8, one as serotype 1 and one as serotype 12. Adeno-associated viruses were only observed in combination with adenoviral particles of the serotype 12 isolate which was derived from a relatively mild outbreak of inclusion body hepatitis. Only the serotype 1 isolate, obtained from the unaffected broiler flock, consistently caused the death of embryos with marked pathological changes. All of the isolates produced basophilic intranuclear inclusion bodies surrounded by clear halos in chicken kidney cell cultures. DNA preparations, obtained from six strains of serotype 8 avian adenovirus (two New Zealand isolates, three Australian isolates and the reference strain HVI) after digestion with the restriction enzymes EcoRI and BamHI, gave electrophoretic patterns showing the New Zealand isolates to be similar to one another and to strain HVI, but quite distinct from the Australian isolates.     

Pathogenicity studies with a strain of fowl adenovirus serotype 8 (VRI-33) in chickens

Experiments were undertaken to study the pathogenesis of VRI-33, a strain of fowl adenovirus serotype 8 isolated from the liver of a broiler chicken with inclusion body hepatitis. A 30% death rate resulted from oral infection of one-day-old specific pathogen free chickens with 10⁶ plaque forming units of VRI-33. Chickens 10, 14, 21 and 28 days of age did not die following infection via natural routes but there were some motalities following infection via parenteral routes. Immunodepression by neonatal cyclophosphamide treatment, followed by infection with VRI-33 via non-parenteral routes, caused varying degrees of hepatitis with basophilic intranuclear inclusion bodies in hepatocytes. The mortality rate of cyclophosphamide-treated, VRI-33 infected chickens, was not significantly altered by post-infection temperature stress. Infection with infectious bursal disease virus, followed by infection with VRI-33 via natural routes at 14 days of age, was not associated with mortalities.     

Avian adenovirus isolated from pigeons affected with inclusion body hepatitis

Avian adenoviruses were isolated from two pigeons affected with inclusion body hepatitis (IBH) by using chicken embryo liver cell cultures. One of the isolates, designated strain S-PL1, replicated in the cell nuclei forming intranuclear inclusion bodies, showed adenovirus-like morphology by electron microscopy, and cross-reacted serologically with strain SR-48 known as serotype 2 of fowl adenovirus. The strain S-PL1 killed day-old chicks by subcutaneous inoculation, and its 50% chicken lethal dose was 10(3.8) plaque forming units per bird. Severe lesions characterized with IBH and pancreatitis, were produced in chicks inoculated with the virus. Intranuclear inclusion bodies were also recognized in the liver, pancreas, kidney, proventriculus, small intestine, and caecum. By indirect immunofluorescence test, intranuclear viral antigens were detected in the liver, pancreas and other tissues.     

Chicken embryo propagation of type I avian adenoviruses

Forty-two clone-purified, cell-culture-propagated type I avian adenoviruses (AAV) representing 11 serotypes and two intermediate strains were evaluated for virus replication (evidenced by embryo death and lesions) resulting from the inoculation of specific-pathogen-free chicken embryos via the chorioallantoic sac or yolk sac. Commonly observed embryonic changes were death, stunting and curling, hepatitis, splenomegaly, congestion and hemorrhage of body parts, and urate formation in the kidneys. Basophilic or eosinophilic intranuclear inclusion bodies characteristic of fowl adenoviruses were observed in hepatocytes. The magnitude and relative uniformity of intra- and interserotypic embryo mortality, gross lesions, and virus titers was greater in embryos inoculated via the yolk sac. This work identifies the yolk sac as a practical and sensitive chicken embryo inoculation route for poultry diagnosticians to employ. It is suggested that the yolk sac may be a reliable alternative to cell culture for the successful isolation of all type I avian adenoviruses.     

Development of an enzyme-linked immunosorbent assay to detect and quantify adenovirus in chicken tissues

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect and quantify avian adenovirus (AAV) in various chicken tissues, including blood. A positive ELISA absorbance value was obtained with suspensions of infected liver tissue that contained less than 100 mean tissue-culture infective doses per gram. A positive correlation was observed between the absorbance values and titer of infectious virus in infected liver tissue. A group-specific antigen common to the 12 serotypes of AAV tested was demonstrated by this ELISA. Because of the high sensitivity and broad-spectrum reactivity, this ELISA could be useful for the study of AAV pathogenesis, for laboratory diagnosis of inclusion body hepatitis irrespective of the serotype of AAV involved, and for screening commercial and specific-pathogen-free flocks for the presence of AAV.     

Structure of inclusion bodies and electron microscopic virus detection in naturally occurring inclusion body hepatitis in chickens]

Electron microscopy was used to examine the liver of chickens with spontaneous inclusion body hepatitis. Eosinophilic inclusion bodies only were established from two flocks, mainly amphophilic from one flock, and primarily basophilic from another two flocks. Eosinophilic inclusion bodies were predominant in broiler chickens with dystrophic fatty degeneration of the liver, while basophile inclusion bodies were recorded primarily from parental or laying-hen chickens with reduced metabolic stress of the liver and more focal necrosis. The eosinophilic inclusion bodies consitsed of a filamentous matrix, with virus particles not safely detectable. The amphophilic inclusion bodies contained parvovirus particles, most likely adenoassociated virus, while the basophilic inclusion bodies inclused parvoviruses or adenoviruses (in flock NO. IV) or adenoviruses only (in flock No. V) in an amorphous chromatin matrix. The presence of parvoviruses in field material was taken to suggest a possible role of those pathogens in inclusion body hepatitis.