肉鸡感染FAdV-4后胸腺组织中部分先天性免疫相关基因的表达变化研究

发布时间：2018-07-27 10:20

【摘要】：禽腺病毒4型(Fowl adenovirus serotype 4,FAdV-4)属于禽腺病毒(Fowl adenovirus,FAV)C型,禽腺病毒属腺病毒科。FAdV-4已经在世界各地禽类中流行并发病,该病不仅对养禽业造成巨大损失,还对世界各地动物生物安全造成巨大危害。近年来,国内外对FAdV-4致病机制的研究主要集中在感染禽类的眼、呼吸系统和消化系统,对免疫系统方面的研究鲜有报道。本试验将FAdV-4人工感染3周龄白羽肉鸡,分别采用HE染色和实时荧光定量PCR法检测了感染后不同时间鸡胸腺组织病理损伤,胸腺中病毒载量及免疫相关基因mRNA的表达变化。获得结果如下:1、将FAdV-4(103 EID50/mL)人工感染非免健康白羽肉鸡,于感染后不同时间采集胸腺组织。感染鸡于感染后第3天(3 dpi)出现明显临床症状,在4 dpi部分感染鸡出现死亡,7 dpi时症状逐渐减轻,15 dpi基本恢复健康。HE染色观察表明:在4 dpi与5dpi胸腺组织出现明显的胸腺萎缩,且出现明显的出血、充血和局部组织坏死等病变,7dpi后病变损伤逐渐减轻。以上结果表明成功建立了FAdV-4感染鸡模型。2、通过荧光定量RT-PCR法对FAdV-4人工感染鸡胸腺组织中病毒载量及免疫相关基因mRNA的检测表明:最早在1dpi即可在胸腺中检出FAdV-4,在4 dpi病毒水平达到最高,随后逐渐降低。TLR3/7及MDA5基因的mRNA表达水平均在4dpi达到峰值随后逐渐降低,其中MDA5在4dpi上调水平较未感染对照组差异极显著(P0.001);IL-2、IFN-α、IFN-β在胸腺组织中表达水平均在4 dpi达到峰值,上调倍数分别为31.79、4.59、3.75;胸腺组织中IL-4、IL-10表达水平分别在3 dpi、5 dpi达到峰值,随后逐渐下降;TGF-β1、TGF-β2基因的mRNA表达水平都在4dpi达到峰值,随后逐渐下降。以上结果表明FAdV-4感染鸡胸腺中天然免疫相关基因表达变化趋势与病毒增殖规律相似。本研究结果表明,人工感染FAdV-4能在鸡胸腺组织中存活复制,并且能够引起肉鸡胸腺出现严重病理损伤,同时FAdV-4引起感染鸡胸腺组织中免疫相关基因的表达变化。研究结果对全面了解FAdV-4感染对肉鸡胸腺免疫功能影响及其防治提供实验依据。
[Abstract]:Avian adenovirus type 4 (Fowl adenovirus serotype 4) belongs to Fowl adenovirus type C (Fowl adenovirus.FAdV-4) has been prevalent and developed in poultry all over the world, which not only causes great loss to poultry industry. It also does great harm to animal biosafety around the world. In recent years, researches on the pathogenesis of FAdV-4 mainly focus on the eyes, respiratory system and digestive system of infected birds, but there are few reports on the immune system. Three weeks old white broilers were artificially infected with FAdV-4. The pathological changes of thymus tissues, the viral load and the expression of immune-related gene mRNA in thymus were detected by HE staining and real-time fluorescence quantitative PCR. The results were as follows: 1. FAdV-4 (103 EID50/mL) was artificially infected with non-immune healthy white broilers and thymus tissues were collected at different time after infection. On the 3rd day (3 dpi) after infection, obvious clinical symptoms appeared in infected chickens, and after 4 dpi partial infected chickens died for 7 dpi, the symptoms gradually alleviated and recovered to health for 15 dpi. The results of HE staining showed that the thymus atrophy appeared in 4 dpi and 5dpi thymus tissues. There were obvious bleeding, hyperemia, local tissue necrosis and other lesions after 7 dpi lesion injury gradually reduced. The results showed that the chicken model of FAdV-4 infection was successfully established. The detection of viral load and immune-related gene mRNA in FAdV-4 artificially infected chicken thymus tissue by fluorescence quantitative RT-PCR method showed that FAdV-4 could be detected in the thymus at the earliest in 1dpi, and FAdV-4 could be detected in the thymus of FAdV-4 artificially infected chicken. (4) the level of dpi virus reached the highest level, The level of mRNA expression of. TLR3 / 7 and MDA5 gene reached the peak level in 4dpi and then decreased gradually. The up-regulation level of MDA5 in 4dpi was significantly higher than that in uninfected control group (P0. 001). The level of IFN- 尾 expression in thymus tissues reached the peak in 4 dpi. The upregulation times were 31.79 ~ 4.59 ~ 3.75, respectively, and the expression of IL-4 ~ + IL-10 reached its peak at 3 d ~ (5) dpi, then decreased gradually. The mRNA expression level of TGF- 尾 _ (1) TGF- 尾 _ 2 gene reached its peak in 4dpi and then decreased gradually. These results suggest that the expression trend of innate immune-related genes in chicken thymus infected with FAdV-4 is similar to that of virus proliferation. The results showed that artificial infection of FAdV-4 could survive and replicate in chicken thymus and induce serious pathological damage in broiler thymus. Meanwhile, FAdV-4 could induce the expression of immune-related genes in infected chicken thymus. The results provide experimental basis for understanding the effect of FAdV-4 infection on thymus immune function and its prevention and treatment in broilers.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.31

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