应用原位PCR对鸡传染法氏囊病病毒早期侵染过程的研究

人工接种35日龄SPF鸡传染法氏囊病病毒,间隔不同时间采集法氏囊、胸腺、脾、盲肠扁桃体、肾、肝和腿肌进行了原位PCR检测,同时观察各组织病理变化。结果无论是H毒株还是Ts毒株,接种后4h肝、肾、脾等组织中即出现明显的阳性信号,Ts毒株4hPI从胸腺、盲肠扁桃体、腿肌等组织中也检出了病毒基因序列。接种H毒株和Ts毒株后28h和40h,法氏囊淋巴细胞开始出现变性、坏死。在阳性信号检出的时间上,法氏囊较肝、肾、脾等组织是滞后的。H毒株较Ts毒株在早期对法氏囊具有更强的侵染能力。     

PCR在鸭瘟病诊断和免疫及致病机理研究中的初步应用

根据GenBank文献,应用Oligo 6.0分析软件合成了用于扩增鸭瘟病毒(duck plague virus,DPV)EcoRⅠ765bp片段的1对引物,上游引物(P1)位于EcoR Ⅰ片段的246～266nt,下游引物(P2)位于EcoRⅠ片段的727～744nt,以DPV CHa株DNA为模板,筛选PCR最佳反应条件,建立了检测DPV的PCR方法.应用该方法对强毒株DPV鸭胚培养物和弱毒株的鸡胚培养物进行扩增,均可获得498bp的DNA片段.而对正常鸭(鸡)胚和鸡马立克氏病毒、鸡传染性喉气管炎病毒、鸭病毒性肝炎病毒、多杀性巴氏杆菌、大肠杆菌、鸭副伤寒沙门氏菌和鸭疫里默氏杆菌培养物进行检测,结果均呈阳性.扩增产物测序结果与文献报道一致,证明了PCR方法的特异性.对DPV CHa株鸡胚毒提取物DNA进行检测,其最低检出量为10fg.用病毒分离、Dot-ELISA和PCR三种方法分别检测1990～2002年期间送检的临床样品,对所获得的结果进行χ2分析,证明PCR检出率明显高于前2种方法.CHa株免疫雏鸭后对血液、心、肝、脾、肺、肾、十二指肠、直肠、法氏囊、胸腺、胰腺、延脑、大脑、小脑、舌头、肌肉、骨髓、食道共18种组织和粪便进行PCR检测,结果表明:①皮下接种4h后,心、肝、脾、肾、法氏囊、胸腺、胰腺、延脑、大脑和小脑为阳性,8h后18种组织和粪便均为阳性;②口服接种4h后舌头和食道为阳性,8h后,心、肝、脾、肾、胸腺、胰腺、延脑、大脑、小脑、舌头、食道和血液均为阳性;③滴鼻接种4h后无阳性组织,8h后检测心、肝、脾、肾、胸腺、延脑、大脑、小脑、舌头、食道和血液,均为阳性;④三种途径免疫的鸭,于12h至第21天均检测出DPV DNA.DPV强毒SC1株人工感染成年鸭2h后,即能从脑、肝、脾、法氏囊和胸腺中检出DPV DNA.12h后和死亡鸭的心、肝、脾、肺、肾、十二指肠、直肠、法氏囊、胸腺、胰腺、脑、胸肌、食道、腺胃、血液、舌头、皮肤、骨髓等组织器官和口腔分泌物及粪便中,均检测到DPV的DNA.该研究为阐明鸭瘟病毒在体内分布提供了重要的数据.     

PCR在鸭瘟临床诊断和免疫及致病机理研究中的初步应用

根据GenBank文献,应用Oligo 6．0分析软件合成了用于扩增鸭瘟病毒(duck plague virus,DPV)EcoR Ⅰ 765bp片段的1对引物,上游引物(P1)位于EcoR Ⅰ片段的246～266nt,下游引物(P2)位于EcoR Ⅰ片段的727～744nt,以DPV CHa株DNA为模板,筛选PCR最佳反应条件,建立了检测DPV的PCR方法。应用该方法对强毒株DPV鸭胚培养物和弱毒株的鸡胚培养物进行扩增,均可获得498bp的DNA片段。而对正常鸭(鸡)胚和鸡马立克氏病毒、鸡传染性喉气管炎病毒、鸭病毒性肝炎病毒、多杀性巴氏杆菌、大肠杆菌、鸭副伤寒沙门氏菌和鸭疫里默氏杆菌培养物进行检测,结果均呈阳性。扩增产物测序结果与文献报道一致,证明了PCR方法的特异性。对DPV CHa株鸡胚毒提取物DNA进行检测,其最低检出量为10fg。用病毒分离、Dot—ELISA和PCR三种方法分别检测1990～2002年期间送检的临床样品,对所获得的结果进行χ^2分析,证明PCR检出率明显高于前2种方法。CHa株免疫雏鸭后对血液、心、肝、脾、肺、肾、十二指肠、直肠、法氏囊、胸腺、胰腺、延脑、大脑、小脑、舌头、肌肉、骨髓、食道共18种组织和粪便进行PCR检测,结果表明：①皮下接种4h后,心、肝、脾、肾、法氏囊、胸腺、胰腺、延脑、大脑和小脑为阳性,8h后18种组织和粪便均为阳性;②口服接种4h后舌头和食道为阳性,8h后,心、肝、脾、肾、胸腺、胰腺、延脑、大脑、小脑、舌头、食道和血液均为阳性;③滴鼻接种4h后无阳性组织,8h后检测心、肝、脾、肾、胸腺、延脑、大脑、小脑、舌头、食道和血液,均为阳性;④三种途径免疫的鸭,于12h至第21天均检测出DPV DNA。DPV强毒SC1株人工感染成年鸭2h后,即能从脑、肝、脾、法氏囊和胸腺中检出DPV DNA。12h后和死亡鸭的心、肝、脾、肺、肾、十二指肠、直肠、法氏囊、胸腺、胰腺、脑、胸肌、食道、腺胃、血液、舌头、皮肤、骨髓等组织器官和口腔分泌物及粪便中,均检测到DPV的DNA。该研究为阐明鸭瘟病毒在体内分布提供了重要的数据。     

流行性出血热病毒感染金黄地鼠的实验研究

本文证明金黄地鼠对于流行性出血热病毒包括家鼠型和野鼠型毒株均敏感。除口饲外,多途径接种均可使地鼠感染。流行性出血热病毒浙5株10001D_(50)接种3周龄地鼠,五天后即可在肺、脾、肠、肝等多种组织中检出病害抗原,至8—10天达最峰,其中肺最强,抗原持续时间至少50天。接种病毒后10天左右,可在血中检出抗体,后缓慢上升,至第50天达1:1280—1:5120。乳鼠感染EHF病毒后可引起全身播散性感染,病毒抗原可在全身软组织中找到。乳鼠感染强毒株可发病致死,但幼鼠和成鼠不表现症状为健康带毒者。     

秦岭地区大林姬鼠的酯酶和苹果酸脱氢酶同工酶的研究

用聚丙烯酰胺凝胶等电聚焦电泳分析了大林姬鼠(Apodemus peninsulae)心、肝、脾、肾和腿肌的 α-酯酶,β-酯酶和苹果酸脱氢酶同工酶。结果表明3种同工酶的活性在5种器官组织中均有明显差异,其中以肝组织的酯酶活性最高,不同器官组织的酶谱也有明显差别,如脾的β一酯酶仅有B区带,同一器官组织通常以α-酯酶活性高于β-酯酶。苹果酸脱氢酶在碱性溶液中染色,肝组织有明显的AB医。心肌与腿肌的苹果酸脱氢酶活性略高于其他组织。     

禽流感H5N1型病毒对沙鼠致病性的初步研究

[目的]观察禽流感H5N1型病毒对沙鼠的致病性;[方法]在生物安全三级实验室,将禽流感H5N1型病毒通过滴鼻接种乙醚麻醉后沙鼠,观察14天,记录沙鼠的体温体重、临床症状、病理变化、病毒分离及抗体变化;[结果]沙鼠感染后发病主要表现在第2天至第6天,攻毒组沙鼠出现反应迟钝、皱毛、弓背、食欲下降、呼吸急促、打堆等症状,攻毒组沙鼠的体温降低和体重减轻,死亡率为44%,在第8天检出抗体,主要病理变化表现为肺出现严重淤血、水肿、出血,镜下可见肺间质充血,血管周围炎性细胞浸润,肝和胸腺淤血,肾出血,肾小管变形。     

免疫组化法检测美国青蛙组织中的蛙虹彩病毒

分不同时期,收集经人工感染了蛙病毒的样蛙,取其心、肺、肾、肠脾、肝六种组织,并通过免疫组化方法进行检测。结果分别在感染了病毒3d、9d、11d后的幼蛙这六种组织中,由表及里观察到了深色的阳性信号,从而测定了病毒在入侵组织中的存在部位,其中,肺和肠组织中阳性信号最强,呈灶性分布,其余四种组织中的阳性信号则呈散性分布。在未注射病毒的幼蛙阴性对照组六种组织设计中没有检测到阳性信号。     

鸭病毒性肠炎病毒强毒株的形态发生学与超微病理学研究

应用透射电镜和超薄切片技术,研究鸭病毒性肠炎病毒(duck enteritis virus,DEV)CH强毒株人工感染成年鸭后,病毒在宿主细胞内的形态发生及各组织器官的超微结构变化.结果表明,感染后不同时间剖杀及发病后死亡鸭的肝、肠、脾、胸腺、法氏囊等组织器官中,均观察到典型的疱疹病毒粒子.病毒主要的靶细胞为淋巴细胞、网状内皮细胞、成纤维细胞、巨噬细胞、血管内皮细胞、肠道上皮细胞、肠道平滑肌细胞和肝细胞等.DEV的核衣壳有空心型、致密核心型、双环型和内壁附有颗粒型四种形态,存在胞核和胞浆两种装配方式.病毒核衣壳可在核内获得皮层,通过核内膜获得囊膜成为成熟病毒;也可通过内外核膜进入胞浆,在其中获得皮层,然后在各种质膜上获得囊膜,最后成熟病毒释放到细胞外.伴随着病毒的复制、装配和成熟,细胞中出现多种核内和胞浆包涵体、核内致密病毒核酸颗粒、微管和中空短管以及胞浆内膜包裹的电子致密小体、双层管等病毒相关结构.超微研究表明,组织细胞有坏死和凋亡两种变化.坏死细胞肿胀甚至破裂,线粒体肿胀空泡化,粗面内质网扩张,核糖体脱落,有的细胞器甚至完全崩解,染色质或固缩或溶解.凋亡细胞则染色质聚集,胞浆凝聚深染,细胞膜上有大量空泡,并有凋亡小体形成.细胞坏死与凋亡往往同时存在,疾病发生过程中,脾、胸腺、法氏囊以及小肠固有层中的淋巴细胞凋亡数量明显增多.     

秦岭地区大林姬鼠(Apodemus peninsulae)的酯酶和苹果酸脱氢酶同工酶的研究

用聚丙烯酰胺凝胶等电聚焦电泳分析了大林姬属(Apodemus peninsulae)心、肝、脾、肾和腿肌的α—酯酶、β—酯酶和苹果酸脱氢酶同工酶。结果表明3种同工酶的活性在5种器官组织中均有明显差异,其中以肝组织的酯酶活性最高;不同器官组织的酶谱也有明显差别,如脾的β—酯酶仅有B区带;同一器官组织通常以α—酯酶活性高于β—酯酶。苹果酸脱氢酶在碱性溶液中染色,肝组织有明显的AB区。心肌与腿肌的苹果酸脱氢酶活性略高于其他组织。     

H5N1禽流感病毒对C57BL／6小鼠的致病性研究

本试验利用虎源H5N1禽流感病毒对6～8周龄雌性C57BL/6小鼠进行滴鼻感染,观测小鼠临床症状和组织病理变化,于感染后第3d和第5d每组分别处死3只小鼠,测定肺、脑、脾、肾、肝组织中的病毒含量;并测定该病毒对C57BL/6小鼠的MLD50。结果表明感染小鼠出现精神不振、体重下降、支气管炎和间质性肺炎为主的临床症状和病理变化;测得感染后小鼠肺脏中病毒拷贝数和病毒滴度最高,其次是脑、肾、脾、肝等组织;该病毒对C57BL/6小鼠的MLD50为10-6.5/0.05mL。此研究成功进行了H5N1禽流感病毒对C57BL/6小鼠的感染,可以作为感染模型进行H5N1禽流感病毒的发病机制、疫苗评价、药物筛选等研究。     

Characteristics of bursal T lymphocytes induced by infectious bursal disease virus

Infectious bursal disease virus (IBDV) is an avian lymphotropic virus that causes immunosuppression. When specific-pathogen-free chickens were exposed to a pathogenic strain of IBDV (IM), the virus rapidly destroyed B cells in the bursa of Fabricius. Extensive viral replication was accompanied by an infiltration of T cells in the bursa. We studied the characteristics of intrabursal T lymphocytes in IBDV-infected chickens and examined whether T cells were involved in virus clearance. Flow cytometric analysis of single-cell suspensions of the bursal tissue revealed that T cells were first detectable at 4 days postinoculation (p.i.). At 7 days p.i., 65% of bursal cells were T cells and 7% were B cells. After virus infection, the numbers of bursal T cells expressing activation markers Ia and CD25 were significantly increased (P<0.03). In addition, IBDV-induced bursal T cells produced elevated levels of interleukin-6-like factor and nitric oxide-inducing factor in vitro. Spleen and bursal cells of IBDV-infected chickens had upregulated gamma interferon gene expression in comparison with virus-free chickens. In IBDV-infected chickens, bursal T cells proliferated in vitro upon stimulation with purified IBDV in a dose-dependent manner (P<0.02), whereas virus-specific T-cell expansion was not detected in the spleen. Cyclosporin A treatment, which reduced the number of circulating T cells and compromised T-cell mitogenesis, increased viral burden in the bursae of IBDV-infected chickens. The results suggest that intrabursal T cells and T-cell-mediated responses may be important in viral clearance and promoting recovery from infection.     

Self-assembly of the infectious bursal disease virus capsid protein, rVP2, expressed in insect cells and purification of immunogenic chimeric rVP2H particles by immobilized metal-ion affinity chromatography

A gene encoding a structural protein (VP2) of a local strain (P3009) of infectious bursal disease virus (IBDV) was cloned and expressed using the baculovirus expression system to develop a subunit vaccine against IBDV infection in Taiwan. The expressed rVP2 proteins formed particles of approximately 20-30 nm in diameter. Those particles were partially purified employing sucrose density gradient ultracentrifugation, and the purified particles were recognized by a monoclonal antibody against the VP2 protein of IBDV P3009. To facilitate the purification of the particles, the VP2 protein was engineered to incorporate a metal ion binding site (His)(6 )at its C-terminus. The chimeric rVP2H proteins also formed particles, which could be affinity-purified in one step with immobilized metal ions (Ni(2+)). Particle formation was confirmed by direct observation under the electron microscope. The production level of rVP2H protein was determined to be 20 mg/L in a batch culture of Hi-5 cells by quantifying the concentration of the purified proteins. The chicken protection assay was performed to evaluate the immunogenicity of the rVP2H protein. When susceptible chickens were inoculated with the recombinant rVP2H proteins (40 microg/bird), virus-neutralizing antibodies were induced, thereby conferring a high level of protection against the challenge of a very virulent strain of IBDV. In conclusion, the most significant finding in this work is that both of the expressed rVP2 and rVP2H proteins can form a particulate structure capable of inducing a strong immunological response in a vaccinated chicken.     

Inhibitory effect of Sargassum polysaccharide on oxidative stress induced by infectious bursa disease virus in chicken bursal lymphocytes

The aim of this study was to investigate the inhibitory effect of Sargassum polysaccharide on oxidative stress induced by infectious bursa disease virus (IBDV) in chicken bursal lymphocytes. The levels of IL-1β, IL-8, IL-10, TNF-α, MCP-1, reduced glutathione and reactive oxygen species in chicken bursal lymphocytes treated with IBDV or both IBDV and Sargassum polysaccharide were measured, and the activities of superoxide dimutase and glutathione peroxidase were evaluated. Our results showed that oxidative stress appeared when chicken bursal lymphocytes were incubated with IBDV for 8 h at 100 TCID₅₀. Sargassum polysaccharide inhibited oxidative stress by increasing the amount of reduced glutathione, promoting the activities of superoxide dimutase and glutathione peroxidase and reducing the level of reactive oxygen species. The polysaccharide also raised IL-1β, IL-8, IL-10 and TNF-α levels in cells infected with IBDV. These findings suggest that Sargassum polysaccharide acts against infection by elevating antioxidant capacity and cytokine levels in chicken bursal lymphocytes.     

Apoptotic response of chicken embryonic fibroblast cells to infectious bursal disease virus infections reflects viral pathogenicity

Infectious bursal disease virus (IBDV) induces immunodeficiency in young chickens and apoptosis in chicken embryos. To understand the relation between the viral pathogenesis and the induction of cell death, chicken embryonic fibroblast (CEF) cells were infected with IBDV intermediate (im) and very virulent (vv) strains at different MOIs. The cell viability and DNA fragmentation were evaluated in infected cells. The cellular apoptotic pathway involve was investigated by determining the activities of caspase cascade. The imIBDV strain was replicated well in CEF cells and shown higher viral titers than vvIBDV. Apoptosis changes were observed only in vvIBDV-infected CEF cells at higher MOI 48 h post infection. Efflux of cytochrome c suggests that the intrinsic pathway of the apoptotic process induced by vvIBDV infection independently of virus replication. Prediction of caspase substrates cleavage sites revealed that different IBDV strains have conserved cleavage motif pattern for VP2 and VP5 viral proteins. These findings suggest the pathogenicity of IBDV strains might be involved in the induction of apoptosis in host cells.     

Protective Oral Vaccination against Infectious bursal disease virus Using the Major Viral Antigenic Protein VP2 Produced in Pichia pastoris

Infectious bursal disease virus (IBDV) causes economically important immunosuppressive disease in young chickens. The self-assembling capsid protein (VP2) from IBDV strain IR01 was expressed in Pichia pastoris resulting in the formation of homomeric, 23-nm infectious bursal disease subviral particles (IBD-SVPs) with a yield of 76 mg/l before and 38 mg/l after purification. Anti-IBDV antibodies were detected in chickens injected with purified IBD-SVPs or fed with either purified IBD-SVPs or inactivated P. pastoris cells containing IBD-VP2 (cell-encapsulated). Challenge studies using the heterologous classical IBDV strain (MB3) showed that intramuscular vaccination with 20 µg purified IBD-SVPs conferred full protection, achieved complete virus clearance and prevented bursal damage and atrophy, compared with only 40% protection, 0–10% virus clearance accompanied by severe atrophy and substantial bursal damage in mock-vaccinated and challenge controls. The commercial IBDV vaccine also conferred full protection and achieved complete virus clearance, albeit with partial bursal atrophy. Oral administration of 500 µg purified IBD-SVPs with and without adjuvant conferred 100% protection but achieved only 60% virus clearance with adjuvant and none without it. Moderate bursal damage was observed in both cases but the inclusion of adjuvant resulted in bursal atrophy similar to that observed with live-attenuated vaccine and parenteral administration of 20 µg purified IBD-SVPs. The oral administration of 250 mg P. pastoris cells containing IBD-VP2 resulted in 100% protection with adjuvant and 60% without, accompanied by moderate bursal damage and atrophy in both groups, whereas 25 mg P. pastoris cells containing IBD-VP2 resulted in 90–100% protection with moderate bursal lesions and severe atrophy. Finally, the oral delivery of 50 µg purified IBD-SVPs achieved 40–60% protection with severe bursal lesions and atrophy. Both oral and parenteral administration of yeast-derived IBD-VP2 can therefore induce a specific and protective immune response against IBDV without affecting the growth rate of chickens.     

Determination of virulence of different strains of Listeria monocytogenes and Listeria innocua by oral inoculation of pregnant mice.

A pregnant mouse model was developed to follow the course of infection after peroral inoculation with six different strains of Listeria monocytogenes and one strain of Listeria innocua. Tissues were sampled and analyzed by microbiologic and histologic methods for 5 days postinoculation. In gnotobiotic pregnant BALB/c mice, L. monocytogenes Scott A (SA), serotype 4b, colonized the gastrointestinal tract, translocated to the livers and spleens of mice by day 1 postinoculation, and multiplied in these tissues until day 4. Infection of the placental tissues occurred by days 3 and 4 and was followed by infection of the fetuses. Little damage of colonic and cecal tissues was evident by histologic examination. Livers and spleens showed a cellular immune response; a similar immune response was not detected in the placentas or fetuses. A rough variant of L. monocytogenes SA which was as virulent as the parent strain in mice when injected intraperitoneally was less virulent perorally and did not consistently infect the fetuses. L. monocytogenes ATCC 19113, serotype 3a, did not colonize the gastrointestinal tract, nor was it isolated from any internal tissue. L. monocytogenes strains of serotypes 1/2a and 1/2b behaved like the SA strain in this mouse model. L. innocua colonized the gastrointestinal tract and translocated to the livers and spleens but did not survive in these organs and rapidly disappeared without infecting placental and fetal tissues. In comparison with gnotobiotic mice, conventional pregnant mice inoculated with L. monocytogenes strains showed less consistent infection. These results suggest that the gnotobiotic pregnant mouse is a useful model for detecting differences in virulence relating to colonization, invasiveness, and uteroplacental infection which cannot be detected by intraperitoneal inoculation of mice.     

Determination of virulence of different strains of Listeria monocytogenes and Listeria innocua by oral inoculation of pregnant mice.

A pregnant mouse model was developed to follow the course of infection after peroral inoculation with six different strains of Listeria monocytogenes and one strain of Listeria innocua. Tissues were sampled and analyzed by microbiologic and histologic methods for 5 days postinoculation. In gnotobiotic pregnant BALB/c mice, L. monocytogenes Scott A (SA), serotype 4b, colonized the gastrointestinal tract, translocated to the livers and spleens of mice by day 1 postinoculation, and multiplied in these tissues until day 4. Infection of the placental tissues occurred by days 3 and 4 and was followed by infection of the fetuses. Little damage of colonic and cecal tissues was evident by histologic examination. Livers and spleens showed a cellular immune response; a similar immune response was not detected in the placentas or fetuses. A rough variant of L. monocytogenes SA which was as virulent as the parent strain in mice when injected intraperitoneally was less virulent perorally and did not consistently infect the fetuses. L. monocytogenes ATCC 19113, serotype 3a, did not colonize the gastrointestinal tract, nor was it isolated from any internal tissue. L. monocytogenes strains of serotypes 1/2a and 1/2b behaved like the SA strain in this mouse model. L. innocua colonized the gastrointestinal tract and translocated to the livers and spleens but did not survive in these organs and rapidly disappeared without infecting placental and fetal tissues. In comparison with gnotobiotic mice, conventional pregnant mice inoculated with L. monocytogenes strains showed less consistent infection. These results suggest that the gnotobiotic pregnant mouse is a useful model for detecting differences in virulence relating to colonization, invasiveness, and uteroplacental infection which cannot be detected by intraperitoneal inoculation of mice.     

禽呼肠孤病毒感染对鸡法氏囊及免疫应答的影响

研究LY株禽呼肠孤病毒(ARV)感染1日龄SPF鸡后对法氏囊发育影响,对传染性法氏囊病毒(IBDV)、禽流感病毒(AIV)、新城疫病毒(NDV)疫苗免疫诱发的抗体的影响,及对强毒株IBDV致病作用的影响。结果表明,LY株ARV感染1日龄SPF鸡可引起法氏囊萎缩和部分淋巴细胞减少,但对增重及AIV和NDV疫苗免疫后抗体滴度却没有显著影响。ARV感染可降低弱毒IBDV疫苗免疫后的抗体反应,但对随后IBDV强毒株攻毒的抵抗力却与对照鸡无显著差异。经IBDV弱毒疫苗免疫后,再接种强毒株IBDV,不会引起死亡,但却仍能显著抑制对AIV、NDV疫苗免疫后的抗体滴度。然而,对于1~7日龄经ARV感染的鸡,IBDV强毒的这种免疫抑制作用又显著低于未经ARV感染的对照鸡。