【摘要】：禽腺病毒Ⅰ群血清4型(Fowl adenovirus serotype 4,FAdV-4)病毒感染可引起家禽心包積液綜合征(Hydropericardium syndrome,HPS)。肉雞感染FAdV-4后剖檢病變主要表現(xiàn)為心包腔內(nèi)充盈大量黃色液體、肝臟腫大和壞死。肝臟最典型的病理組織學(xué)變化是肝細胞核內(nèi)出現(xiàn)嗜堿性包涵體。肝臟是FAdV-4主要的靶器官,然而目前關(guān)于FAdV-4對肉雞肝臟炎癥損傷機理的研究鮮有報道。本研究用FAdV-4人工感染3周齡白羽艾維茵肉雞,HE染色觀察病毒感染后肝臟的組織病變;采用real-time PCR檢測不同感染時間點的肝臟病毒載量;應(yīng)用real-time RT-PCR檢測肝臟炎癥相關(guān)基因在不同感染時間的表達變化。目的是揭示肉雞感染FAdV-4致使肝臟炎癥損傷的機理。試驗結(jié)果如下:1.FAdV-4分離株人工感染能引起非免健康白羽艾維茵肉雞發(fā)生心包積液綜合征。用FAdV-4人工感染非免健康肉雞,分別于1 dpi、3 dpi、4 dpi、5 dpi、7 dpi、9 dpi、11dpi、15 dpi頸部放血處死8只感染肉雞,按無菌操作采取肝臟。FAdV-4感染肉雞在4 dpi天出現(xiàn)死亡,5 dpi病雞出現(xiàn)精神萎靡、食欲減退等。剖檢病雞可見心包腔內(nèi)充滿大量黃色液體和肝壞死。病雞的肝臟HE染色可見大量炎性細胞浸潤,肝細胞變性、壞死,肝細胞核內(nèi)嗜堿性包涵體。2.建立了檢測FAdV-4 Hexon基因real-time PCR的方法。參照GenBank中FAdV-4保守序列Hexon設(shè)計引物,建立了檢測FAdV-4 Hexon基因的real-time PCR的方法。并從感染FAd V-4不同時間點肉雞肝臟中均檢測出該病毒,病毒載量在4 dpi達到峰值。3.采用real-time RT-PCR檢測了FAdV-4感染后不同時間點肉雞肝臟中主要炎癥相關(guān)基因mRNA表達變化。IL-2基因在肝臟中極顯著上調(diào)(P0.001),IL-6/IFN-β基因上調(diào);IFN-α基因在感染初期下調(diào),在5 dpi顯著上調(diào)(P0.01);CXCLi1、CXCLi2和CCR5基因都表現(xiàn)上調(diào),其中CXCLi2基因上調(diào)倍數(shù)最高(P0.001)。IL-4和IL-10基因均呈上調(diào)趨勢,其中IL-4基因在第3 dpi,IL-10基因在4 dpi極顯著上調(diào)(P0.001);TGF-β1和TGF-β2基因在1 dpi下調(diào)(P0.01),隨后上調(diào),其中TGF-β2基因在4 dpi顯著上調(diào)(P0.001)。綜上所述,FAdV-4感染白羽艾維茵肉雞后能造成肝臟嚴重病理損傷,引起肝臟組織中炎癥相關(guān)基因mRNA的表達發(fā)生明顯變化。試驗結(jié)果為揭示FAdV-4感染肉雞肝臟組織炎癥損傷機制提供了試驗數(shù)據(jù)。
[Abstract]:Avian adenovirus group I serotype 4 (Fowl adenovirus serotype 4 (FAdV-4) virus infection may cause pericardial effusion syndrome (Hydropericardium syndrome,HPS) in poultry. The pathological changes after FAdV-4 infection in broilers were mainly yellow fluid in pericardial cavity, hepatomegaly and necrosis. The most typical histopathological changes of the liver are basophilic inclusion bodies in the nucleus of the liver. Liver is the main target organ of FAdV-4. However, there are few reports on the mechanism of liver inflammation injury caused by FAdV-4 in broilers. In this study, three weeks old white feather Avian broilers infected with FAdV-4 were used to observe the liver pathological changes by HE staining, and real-time PCR was used to detect the viral load of the liver at different time points. Real-time RT-PCR was used to detect the expression of inflammatory genes in liver at different infection time. The aim of this study was to elucidate the mechanism of liver inflammatory injury caused by FAdV-4 infection in broilers. The results were as follows: artificial infection of 1.FAdV-4 isolate could induce pericardial effusion syndrome in non-immune white feather Avian broilers. Non-immune broilers were artificially infected with FAdV-4, and 8 infected broilers were killed in 1 dpi,3 dpi,4 dpi,5 dpi,7 dpi,9 dpi,11dpi,15 dpi neck bloodletting. FAdV-4 infected broilers died on 4 dpi day, 5 dpi infected broilers suffered from mental retardation and anorexia. A large amount of yellow fluid and liver necrosis were found in the pericardial cavity of the diseased chicken. A large number of inflammatory cell infiltration, hepatocyte degeneration, necrosis and basophilic inclusion bodies were observed in liver HE staining of diseased chicken. A method for detecting real-time PCR of FAdV-4 Hexon gene was established. According to the FAdV-4 conserved sequence Hexon in GenBank, the primers were designed and a method for detecting real-time PCR of FAdV-4 Hexon gene was established. The virus was detected from the liver of broilers infected with FAd V-4 at different time points, and the viral load reached a peak value of 3. 3 at 4 dpi. Real-time RT-PCR was used to detect the changes of mRNA expression of the major inflammatory related genes in the liver of broilers at different time points after FAdV-4 infection. IL-2 gene was significantly up-regulated (P0.001) and IL-6/IFN- 尾 gene was up-regulated in the liver. IFN- 偽 gene was down-regulated at the early stage of infection and significantly up-regulated at 5 dpi (P0.01). Both CXCLi1,CXCLi2 and CCR5 genes were up-regulated, among which CXCLi2 gene had the highest up-regulation (P0.001). Both IL-4 and IL-10 genes were up-regulated, and IL-4 gene was significantly up-regulated at 4 dpi in the 3rd dpi,IL-10 gene (P0.001). TGF- 尾 1 and TGF- 尾 2 genes were down-regulated at 1 dpi (P0.01) and then up-regulated. TGF- 尾 2 gene was significantly up-regulated at 4 dpi (P0.001). To sum up, FAdV-4 infection with Avian broilers can cause serious pathological damage of liver and change the expression of inflammatory related gene mRNA in liver tissue. The results provide experimental data for revealing the mechanism of liver inflammation injury in broilers infected with FAdV-4.
【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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本文編號：2382967