【摘要】：禽流感病毒(Avian Influenza Virus,AIV)能夠感染多種禽類和包括人類在內(nèi)的哺乳類動(dòng)物。自1994年我國(guó)首次報(bào)道從廣東雞群中分離到H9N2亞型AIV以來,該病毒一直在我國(guó)家禽中廣泛流行。雖然該病毒致病力較低,但是能引起家禽的呼吸道癥狀以及產(chǎn)蛋家禽明顯的產(chǎn)蛋下降給我國(guó)家禽養(yǎng)殖業(yè)帶來巨大的經(jīng)濟(jì)損失。水禽尤其是鴨、鵝是AIV天然的貯存庫(kù),在AIV的傳播過程中起到重要的作用。在流感病毒的基因組的八個(gè)片段中,HA基因具有決定致病力、抗原性和宿主范圍等方面的重要作用,而且HA基因最容易發(fā)生變異。因此研究H9N2亞型AIV對(duì)鵝的致病性以及監(jiān)測(cè)其HA基因的變異情況,有非常重要的意義。1.H9亞型AIV的分離鑒定以及HA基因的變異分析本研究匯集了2012~2013年山東不同地區(qū)的疑似感染H9亞型AIV的病例,收集肺臟樣品,分離到25株病毒,經(jīng)鑒定為AIV。利用PCR技術(shù)擴(kuò)增25株AIV分離株的HA基因,測(cè)序,分析其變異情況。結(jié)果顯示:(1)25株AIV分離株的HA基因核苷酸同源性和氨基酸同源性分別在94.5%~100.0%、93.6%~100.0%之間;(2)遺傳進(jìn)化分析結(jié)果表明,25株AIV分離株均屬于歐亞分支中的Y280-like亞分支;(3)AIV的HA蛋白有7~9個(gè)潛在糖基化位點(diǎn),其中218位糖基化位點(diǎn)缺失,145位新增糖基化位點(diǎn);(4)25株AIV分離株HA蛋白裂解位點(diǎn)均為RSSR↓GIF,符合低致病力AIV的特征;(5)25株AIV分離株的受體結(jié)合位點(diǎn)較保守,234位受體結(jié)合位點(diǎn)均為L(zhǎng)(亮氨酸),僅198位受體結(jié)合位點(diǎn)存在變異,具有與哺乳動(dòng)物唾液酸α,2-6受體結(jié)合的特征。2.H9N2亞型AIV對(duì)鵝的致病性研究本研究選用本實(shí)驗(yàn)室分離、鑒定并保存的一株鵝源H9N2亞型AIV(以下稱為g AIV-H9N2)進(jìn)行人工感染試驗(yàn)。將60只2周齡健康鵝隨機(jī)分為靜脈注射組、點(diǎn)眼滴鼻組和對(duì)照組,每組20只,其中靜脈注射組和點(diǎn)眼滴鼻組每只人工接種1 m L g AIV-H9N2增殖尿囊液(ELD50=10-7.5/0.2 m L),對(duì)照組接種等量的無菌生理鹽水。試驗(yàn)組與對(duì)照組分開隔離飼養(yǎng),每天觀察各組試驗(yàn)鵝的發(fā)病情況和臨床表現(xiàn)。在攻毒后第3、6、9、12、15、21、28d采集血液一次,分離血清,用HA-HI方法測(cè)定血清中H9亞型AIV抗體水平,用細(xì)胞因子ELISA方法檢測(cè)試劑盒檢測(cè)血清中IL-2、IL-6、IFN-β、IFN-γ等細(xì)胞因子的表達(dá)水平,同時(shí)測(cè)定血液生化指標(biāo)。在攻毒后第3、6、9、12、15d剖殺每組的鵝3只,觀察剖檢變化;并采集腦、肝臟、肺臟、胰臟、脾臟等組織器官,用4%福爾馬林溶液固定,按常規(guī)方法制備石蠟切片、HE染色,進(jìn)行病理組織學(xué)觀察。結(jié)果顯示:(1)人工感染后靜脈注射組和點(diǎn)眼滴鼻組鵝表現(xiàn)為精神萎頓,采食量下降,飲水減少,排白色稀便,生長(zhǎng)緩慢,呼吸系統(tǒng)癥狀。靜脈注射組較點(diǎn)眼滴鼻組的鵝臨床感染癥狀明顯,試驗(yàn)過程中無死亡。人工感染后第9 d開始,所有鵝均表現(xiàn)耐過,精神狀態(tài)、采食量、飲水均恢復(fù)正常。對(duì)照組鵝均無異常癥狀。(2)人工感染后,第3d,可見靜脈注射組和點(diǎn)眼滴鼻組肺臟出血,心外膜出血。第6d,可見靜脈注射組和點(diǎn)眼滴鼻組腺胃潮紅,肺臟出血。第9d,可見靜脈注射組和點(diǎn)眼滴鼻組均肝臟淤血、出血,脾臟腫大、有壞死點(diǎn),肺臟出血。第12d,可見靜脈注射組和點(diǎn)眼滴鼻組肺臟出血、腦膜出血點(diǎn)。第15d,可見脾臟腫大,肺臟出血。比較發(fā)現(xiàn),靜脈注射組的各種病變均比點(diǎn)眼滴鼻組嚴(yán)重。對(duì)照組雛鴨未見肉眼可見病變。(3)人工感染后,各組鵝的病理組織學(xué)變化如下。肺臟:攻毒組肺臟出血明顯。氣管:攻毒組氣管杯狀細(xì)胞增多,分泌功能增強(qiáng)。腸道:靜脈注射組腸絨毛杯狀細(xì)胞壞死,絨毛斷裂、脫落,點(diǎn)眼滴鼻組病變不明顯。腦:靜脈注射組腦小膠質(zhì)細(xì)胞增生。肝臟:靜脈注射組肝細(xì)胞水腫、壞死,點(diǎn)眼滴鼻組炎性細(xì)胞浸潤(rùn),淤血。胰臟:局部有典型的炎性細(xì)胞聚集。(4)抗體水平測(cè)定:靜脈注射組和點(diǎn)眼滴鼻組的抗體水平都在攻毒后逐漸升高,到達(dá)高峰后降低,可見靜脈注射組抗體水平升高較快,且前期抗體水平值較高。靜脈注射組和點(diǎn)眼滴鼻組最高抗體滴度都是7log2。(5)細(xì)胞因子測(cè)定:各攻毒組的IL-2、IL-6、IFN-β、IFN-γ水平明顯高于對(duì)照組,在攻毒15天后基本恢復(fù)至與對(duì)照組持平。(6)血液生化指標(biāo)變化如下�？偟鞍鬃兓厔�(shì):在攻毒后,靜脈注射組和點(diǎn)眼滴鼻組的總蛋白量不斷升高,至攻毒后第6d,水平達(dá)到最高值,之后逐漸下降;可見靜脈注射組的總蛋白水平高于點(diǎn)眼滴鼻組。球蛋白變化趨勢(shì):基本與總蛋白的變化趨勢(shì)一致。AST/ALT變化趨勢(shì):人工感染后,水平整體下降,靜脈注射組和點(diǎn)眼滴鼻組下降趨勢(shì)一致。肌酐的變化趨勢(shì):人工感染后,靜脈注射組和點(diǎn)眼滴鼻組肌酐水平先升高后降低,在第6d達(dá)到最高值,而后下降;可見靜脈注射組和點(diǎn)眼滴鼻組的變化幅度基本一致,無太大區(qū)別。
[Abstract]:Avian Influenza Virus (AIV) can infect a variety of poultry and mammals including humans. Since the first report of H9N2 subtype AIV isolated from Guangdong chicken flocks in 1994, the virus has been widespread in poultry in China. Although the virulence of the virus is low, it can cause respiratory symptoms in poultry. Waterfowl, especially ducks and geese, are the natural reservoirs of AIV and play an important role in the transmission of AIV. Among the eight genomic fragments of influenza viruses, HA gene is the major determinant of pathogenicity, antigenicity and host range. Therefore, it is very important to study the pathogenicity of H9N2 subtype AIV in geese and to monitor the variation of its HA gene. 1. Isolation and identification of H9 subtype AIV and analysis of variation of HA gene. This study collected suspected cases of H9 subtype AIV from different areas of Shandong Province from 2012 to 2013. The results showed that: (1) The nucleotide homology and amino acid homology of HA gene of 25 AIV isolates were 94.5%-100.0% and 93.6%-100.0% respectively; (2) Genetic evolution analysis showed that 25 AIV isolates had the same nucleotide homology and amino acid homology. The isolates belong to the Y280-like subbranch of Eurasian branch; (3) There are 7-9 potential glycosylation sites in the HA protein of AIV, of which 218 are deleted and 145 are newly added glycosylation sites; (4) The HA protein cleavage sites of 25 AIV isolates are RSSR_GIF, which conform to the characteristics of low pathogenicity AIV; (5) The receptor binding sites of 25 AIV isolates are more than those of 25 AIV isolates. Conservative, 234 receptor binding sites are L (leucine), only 198 receptor binding sites have mutations, with mammalian sialic acid alpha, 2-6 receptor binding characteristics. 2. H9N2 subtype of AIV pathogenicity to goose This study selected our laboratory isolation, identification and preservation of a goose-derived H9N2 subtype of AIV (hereinafter referred to as G AIV-H9N2) for human. Sixty two-week-old healthy geese were randomly divided into intravenous injection group, intraocular drip nose group and control group, with 20 geese in each group. The intravenous injection group and intraocular drip nose group were artificially inoculated with 1 m L AIV-H9N2 proliferative allantoic fluid (ELD50 = 10-7.5/0.2 m L) and the control group was inoculated with the same amount of sterile saline. Serum samples were collected at 3, 6, 9, 12, 15, 21 and 28 days after treatment. The levels of antibodies against H9 subtype AIV in serum were determined by HA-HI method. The levels of cytokines such as IL-2, IL-6, IFN-beta and IFN-gamma in serum were detected by cytokine ELISA kit. Blood biochemical indices were determined. Three geese in each group were killed on the 3rd, 6th, 9th, 12th and 15th day after poisoning to observe the anatomical changes. Brain, liver, lung, pancreas, spleen and other tissues were collected and fixed with 4% formalin solution. Paraffin sections were prepared by routine method and stained with HE for histopathological observation. The intravenous injection group showed more obvious symptoms of clinical infection than the intravenous drip group, and no death occurred during the experiment. No abnormal symptoms were found in the control group. (2) On the 3rd day after artificial infection, pulmonary hemorrhage, epicardial hemorrhage were observed in the intravenous injection group and the intraocular drip nose group. On the 6th day, glandular stomach flushing and pulmonary hemorrhage were observed in the intravenous injection group and the intraocular drip nose group. On the 12th day, pulmonary hemorrhage and meningeal hemorrhage were observed in the intravenous injection group and the eye dropping nose group. On the 15th day, splenomegaly and pulmonary hemorrhage were observed. Comparing with the eye dropping nose group, all kinds of pathological changes in the intravenous injection group were more serious than those in the eye dropping nose group. Trachea: The goblet cells increased and secretory function was enhanced in the treatment group. Intestinal tract: The goblet cells of intestinal villi necrosis, villi rupture, exfoliation were not obvious in the intravenous injection group. Cerebral microglia proliferation was observed in the intravenous injection group. Pancreas: There were typical inflammatory cell aggregation. (4) Antibody level: The antibody level of intravenous injection group and nose dropping group increased gradually after poisoning, and decreased after reaching the peak. The antibody level of intravenous injection group increased faster, and the pre-antibody level was higher. The levels of IL-2, IL-6, IFN-beta and IFN-gamma in each treatment group were significantly higher than those in the control group, and recovered to the same level after 15 days of treatment. (6) The changes of blood biochemical indexes were as follows. The total protein level of the intravenous injection group was higher than that of the intraocular drip group. The change trend of globulin was basically consistent with that of the total protein. Change trend: After artificial infection, the creatinine level of intravenous injection group and intraocular drip nose group increased first, then decreased, reached the highest value on the 6th day, and then decreased; it can be seen that the changes of intravenous injection group and intraocular drip nose group were basically the same, without much difference.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.33

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本文編號(hào)：2239789