【作者】 刘玉锋；

【导师】 佘锐萍；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2004， 博士

【摘要】 本研究采用组织病理学、超微病理学、组织细胞化学、免疫组织化学、TUNEL技术、ELISA等相结合的方法，对不同毒力IBDV毒株--B87株(疫苗毒)、BC6／85株(强毒株)、SNJ93株(超强毒株)感染雏鸡后的致病特性与免疫学特性进行了全面系统的研究。结果如下： 1．SNJ93株感染后，不仅引起SPF鸡免疫器官严重损伤，而且还引起实质器官出现明显的病理变化。BC6／85株引起的免疫器官损伤较SNJ93株稍轻，只引起肾脏和腺胃—过性的病变，而B87株不引起免疫器官和实质器官损伤。 2．BC6／85株和SNJ93株感染SPF雏鸡后3天，法氏囊指数高于对照组，差异显著(P＜0.05)，感染后5～14天，法氏囊指数明显低于对照组(P＜0.01)，脾脏指数在5日内明显高于对照组(P＜0.01)，胸腺指数在9日内明显低于对照组(P＜0.01)，B87株接种组法氏囊指数和对照组差异不显著(P＞0.05)；脾脏指数在5日内高于对照组(P＜0.05)，但低于SNJ93株与BC6／85株组，差异显著(P＜0.05)；胸腺指数在感染后5日内较BC6／85株攻毒组以及SNJ93株攻毒组高，差异显著(P＜0.05)。 3．ELISA试验表明：BC6／85株与SNJ93株在感染后3～14天内的血清抗体滴度远高于对照组(P＜0.01)，且在14天时BC6／85株比SNJ93株低(P＜0.05)，B87株接种组的血清抗体滴度在3～9天内显著高于对照组(P＜0.01)，在3～7天内低于BC6／85株与SNJ93株攻毒组，差异极显著(P＜0.01)。 4．免疫组织化学观察结果表明，免疫器官中CD4~+、CD8~+T细胞数量较对照组明显升高，从阳性反应强度和持续时间上看，SNJ93株＞BC6／85株＞B87株，BC6／85株与SNJ93株攻毒组各免疫器官中CD8~+T细胞比CD4~+T细胞阳性反应强度明显，B87株接种组二者差别不明显。 5．应用H.E.染色和免疫组化染色方法发现，B87株接种组十二指肠、空肠和回肠中的IEL及sIgA阳性物质较对照组高，差异显著或极显著(P＜0.01或P＜0.05)：SNJ93株与BC6／85株攻毒组十二指肠、空肠和回肠中的IEL及sIgA阳性物质低于对照组，差异显著或极显著(P＜0.01或P＜0.05)。 6．应用免疫组化染色对P物质，VIP两种脑肠肽类物质在实验鸡和对照鸡肠道及免疫器官中的分布观察结果表明：B87株接种组在感染后第3天十二指肠中P物质、VIP含量与对照组均无差异，而感染后第5天均较对照组升高。空肠、盲肠扁桃体、法氏囊、脾脏中这两种物质含量均高于对照组且差异显著(法氏囊中VIP的表达量在第3天与对照组无差异)。SNJ93株和BC6／85株在攻毒后3～5天十二指肠、空肠、盲肠扁桃体、脾脏、胸腺中P物质和VIP的含量较对照组明显升高，差异极其显著(P＜0.01)。 7．利用电镜超薄切片技术对SNJ93株感染鸡的组织器官超微结构观察发现，盲肠扁桃体和肾脏崩解组织中有晶格状排列的病毒粒子，在法氏囊淋巴细胞中观察不到病毒，但却能观察到大量的凋亡小体。TUNEL实验结果表明SNJ93株感染后1～7天可见到大量凋亡的淋巴细胞，其中感染后3～5天最多。BC6／85株攻毒组只在感染后3～5天可见到大量凋亡的淋巴细胞，B87株在接种后1～9天内与对照组差别不显著。 8．细胞凋亡调控蛋白Bcl-2和Bax免疫组织化学观察发现，不同毒力的IBDV感染鸡法氏囊、中国农业人学博_l学位论文中文摘要绝口里里旦口口典里旦口目绝口里旦脾脏、胸腺的淋巴细胞中Bcl一2和Bax蛋白表达有差异。B87株接种组上述三大免疫器官淋巴细胞中这两种蛋白未见表达。SNJ93株攻毒组法氏囊和脾脏淋巴细胞中感染后第2天即有大量Bax蛋白表达，且在感染后3一4天表达量最高，第5天减低。Bd一2蛋白表达起始时间及高峰期与Bax蛋白一致，只是表达时间较Bax长。BC6/85株攻毒组法氏囊淋巴细胞中这两种蛋白只在攻毒后4一5大表达，脾脏淋巴细胞中只在攻毒后第4天表达。 上述实验结果表明，不同毒力的IBDV毒株对鸡体致病性明显不同，实质器官的病变程度与工BDV的毒力呈正相关;超强毒与强毒株虽能引起较强的全身体液和细胞免疫，但局部粘膜免疫受到严重抑制，而疫苗毒可同时激发全身和肠粘膜局部免疫反应;P物质和血管活性肠肤等脑肠肤类物质在工BDV感染后的全身及肠道粘膜局部免疫中有重要的作用;细胞凋亡在法氏囊炎病毒致病机制中起着重要的作用，Bc卜2/Bax能准确反映淋巴细胞的凋亡程度。更多还原

【Abstract】 Using histopathological, electron microscopic, histo-chemical, immuno-histochemical, TUNEL, ELISA methods, Pathogenic and immunological characteristics of various virulent strains of infectious bursal disease virus (IBDV)-B87 strain(attenuted vaccine),BC6/85 strain (virulentIBDV,vIBDV),SNJ93 strain (very virulent IBDV,wIBDV) were observed. The results showed :1.SNJ93 strain caused serious damage not only in immunological organs but also in non-immunological organs. The pathological change of immunological organs of chickens infected with BC6/85 strain was less serious than those infected with SNJ93 strain, and only lesion of proventriculus and kidney were seen in non-immunological organs. B87 strain caused no lesion in immunological organs and non-immunological organs.2.After SPF chickens infected with BC6/85 and SNJ93 strains,the index of bursa was higher than that of the control group(p<0.05) on the third day post infection(PI), but significantly lower from the fifth to fourteenth day PI(p<0.01); The index of spleen was significantly higher than that of the control group from the third to fifth day PI(p<0.01); The index of thymus was significantly lower than that of the control group from the third to ninth day PI (p<0.01). The index of bursa had no significant difference between the B87 strain vaccinal group and control group(p>0.05). The index of spleen of B87 strain vaccinal group was higher than the control group(p<0.05) ,but lower than that of group infected with the SNJ93 or BC6/85 strain from the third to fifth day PI. The index of thymus of B87 strain vaccinal group was higher than that of groups infected with the BC6/85 or SNJ93 strain (p<0.05).3.ELISA method showed the antibody titers of groups infected with the BC6/85 or SNJ93 strain were higher than that of the control group from third to fourteenth day PI (p<0.01), and the former was lower than the latter(p<0.05). the antibody titers of B87 strain vaccinal group were greatly higher than that of the control group from the third to ninth day PI (p<0.01), and lower than groups infected with the BC6/85 or SNJ93 strain from third to seventh day PI (pO.Ol).4.The result of immuno-histochemistrical method showed: the number of CD4+and CD8+T cells of immunological organs in the experimental groups were more than that of the control group. The intensity and durative time of positive reaction were:SNJ93> BC6/85> B87. This positive reaction of CD8+ T cells was more obvious than CD4+ T cells in groups infected with the BC6/85 or SNJ93 strain.5.The result of H.E. and immuno-histochemistrial stain showed the number of IEL and positive slgA cells in duodenum, jejunum and cecal tonsil of B87 strain vaccinal group were more than those of the control group, but which of group infected with the BC6/85 or SNJ93 strain were less than that of the control group6. Substance P and vasoactive intestinal polypeptide (VIP) in duodenum had no difference between the B87 strain vaccinal group and the control group on the third day PI, but they were higher than the control group on the fifth day PI. Substance P and VIP were significantly higher than that of the controlgroup in jejunum, cecal tonsil, bursa and spleen from the third to fifth day PI . After SPF chickens infected with SNJ93 and BC6/85 third to fifth day PI, the substance P and VIP in duodenum, jejunum, cecal tonsil, spleen and thymus were significantly higher than the control group.7.Ultrastructurally, the numerous virus were observed in the cecal tonsil and kidney of groups infected with SNJ93 strain. And apoptotic bodies were observed in the lymhocytes of bursa. TUNEL method showed numerous apoptotic lymphocytes were seen in bursa of chickens infected with SNJ93 from first to seventh day, especially from the third to fifth day PI. Numerous apoptotic lymphocytes were seen in the group infected with BC6/85 strain only from the third to fifth day. But there were no difference between the B87 strain vaccinal group and the control group from first to ninth更多还原