【摘要】：禽腺病毒4型(Fowl adenovirus serotype 4,FAdV-4)屬于禽腺病毒(Fowl adenovirus,FAV)C型,禽腺病毒屬腺病毒科。FAdV-4已經(jīng)在世界各地禽類中流行并發(fā)病,該病不僅對養(yǎng)禽業(yè)造成巨大損失,還對世界各地動物生物安全造成巨大危害。近年來,國內(nèi)外對FAdV-4致病機(jī)制的研究主要集中在感染禽類的眼、呼吸系統(tǒng)和消化系統(tǒng),對免疫系統(tǒng)方面的研究鮮有報道。本試驗將FAdV-4人工感染3周齡白羽肉雞,分別采用HE染色和實時熒光定量PCR法檢測了感染后不同時間雞胸腺組織病理損傷,胸腺中病毒載量及免疫相關(guān)基因mRNA的表達(dá)變化。獲得結(jié)果如下:1、將FAdV-4(103 EID50/mL)人工感染非免健康白羽肉雞,于感染后不同時間采集胸腺組織。感染雞于感染后第3天(3 dpi)出現(xiàn)明顯臨床癥狀,在4 dpi部分感染雞出現(xiàn)死亡,7 dpi時癥狀逐漸減輕,15 dpi基本恢復(fù)健康。HE染色觀察表明:在4 dpi與5dpi胸腺組織出現(xiàn)明顯的胸腺萎縮,且出現(xiàn)明顯的出血、充血和局部組織壞死等病變,7dpi后病變損傷逐漸減輕。以上結(jié)果表明成功建立了FAdV-4感染雞模型。2、通過熒光定量RT-PCR法對FAdV-4人工感染雞胸腺組織中病毒載量及免疫相關(guān)基因mRNA的檢測表明:最早在1dpi即可在胸腺中檢出FAdV-4,在4 dpi病毒水平達(dá)到最高,隨后逐漸降低。TLR3/7及MDA5基因的mRNA表達(dá)水平均在4dpi達(dá)到峰值隨后逐漸降低,其中MDA5在4dpi上調(diào)水平較未感染對照組差異極顯著(P0.001);IL-2、IFN-α、IFN-β在胸腺組織中表達(dá)水平均在4 dpi達(dá)到峰值,上調(diào)倍數(shù)分別為31.79、4.59、3.75;胸腺組織中IL-4、IL-10表達(dá)水平分別在3 dpi、5 dpi達(dá)到峰值,隨后逐漸下降;TGF-β1、TGF-β2基因的mRNA表達(dá)水平都在4dpi達(dá)到峰值,隨后逐漸下降。以上結(jié)果表明FAdV-4感染雞胸腺中天然免疫相關(guān)基因表達(dá)變化趨勢與病毒增殖規(guī)律相似。本研究結(jié)果表明,人工感染FAdV-4能在雞胸腺組織中存活復(fù)制,并且能夠引起肉雞胸腺出現(xiàn)嚴(yán)重病理損傷,同時FAdV-4引起感染雞胸腺組織中免疫相關(guān)基因的表達(dá)變化。研究結(jié)果對全面了解FAdV-4感染對肉雞胸腺免疫功能影響及其防治提供實驗依據(jù)。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.31

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