【摘要】：H9亞型禽流感是目前危害我國養(yǎng)禽業(yè)的主要AIV亞型,以引起病雞體溫升高和呼吸道病變,成年雞產(chǎn)蛋量下降,畸形蛋增多為主要特征。近年來,有研究表明在進行H9亞型禽流感病毒免疫后,仍不斷有疑似病例發(fā)生,且極易與新城疫發(fā)生混合感染,造成經(jīng)濟損失的同時給防疫安排工作帶來了很大的挑戰(zhàn)。因此,將禽流感和新城疫病毒進行聯(lián)合免疫,以防止交叉感染,提高免疫效率,成為如今規(guī)�；B(yǎng)殖場免疫的重要手段。本研究從疑似低致病性禽流感發(fā)病雞場采集病料,分離鑒定出H9亞型禽流感病毒株XY-14株。將此毒株滅活后與新城疫Ⅳ系進行聯(lián)合免疫,以不同免疫方式產(chǎn)生抗體水平的變化為研究重點,了解此毒株與新城疫病毒聯(lián)合免疫產(chǎn)生抗體的快慢與持續(xù)期。本研究主要包括以下幾個方面:1、2014年從信陽雞場采集疑似低致病性禽流感發(fā)病的病料,接種SPF雞胚,分離培養(yǎng)病毒。通過HA(血凝試驗)、HI(血凝抑制試驗)和RT-PCR方法初步篩到1株H9亞型禽流感病毒。將病毒進行終點有限稀釋克隆純化后50倍濃縮蔗糖密度梯度離心提純,取純化好的病毒用禽流感通用引物和H9亞型禽流感通用引物進行擴增,在大約229bp和730bp位置得到明亮的條帶,與預期大小一致。使用特異性引物擴增HA、NA、NP、M、NS基因片段,分別在1740bp、1470bp、1565bp、1027bp和890bp位置出現(xiàn)明亮的條帶,與預期大小一致,因此可確定該毒株為H9亞型。將此毒株命名為A/Chicken/XinYang/XY-14/2014(H9 Subtype),簡稱為XY-14株。2、XY-14株的HA效價為10log2,HI H9效價為10log2,然后用禽流感病毒H9亞型血清進行中和試驗,收集的尿囊液沒有血凝性。3、對該毒株進行毒力測定,做ICPI(1日齡雞腦內(nèi)接種致病指數(shù)統(tǒng)計)ICPI=0.18;和IVPI(雞靜脈接種致病指數(shù))IVPI=0;說明XY-14株屬于弱毒株。4、將XY-14株病毒進行滅活處理,單獨免疫雞,第3周抗體滴度達到峰值12log2,持續(xù)直到第22周抗體水平仍達到10.33log2。結(jié)果表明該滅活毒單獨免疫,能夠使雞產(chǎn)生抗體,且能以很高的抗體滴度提供持續(xù)數(shù)周;與新城疫Ⅳ系苗聯(lián)合免疫0.5mL/羽,第2周便產(chǎn)生ND和AIV H9抗體;ND抗體在第3周達到峰值11log2,持續(xù)到22周仍有6log2的抗體水平;AIV H9抗體水平峰值于第4周出現(xiàn),高達11.33log2。NDⅣ系滴鼻點眼同時再以0.3mL/羽進行聯(lián)合毒株免疫時,不但能同時產(chǎn)生抗兩種病毒的抗體,還能顯著提高ND抗體滴度,但不對抗H9 HI水平產(chǎn)生影響,說明聯(lián)合免疫的兩種病毒之間互不干擾;聯(lián)合免疫0.3mL/羽,同時NDⅣ系滴鼻點眼,在本實驗中為較適免疫方式。
[Abstract]:H9 subtype avian influenza is the main AIV subtype that endangers the poultry industry in China at present. It is characterized by the increase of body temperature and respiratory tract, the decrease of egg production and the increase of abnormal eggs in adult chickens. In recent years, some studies have shown that after immunization with H9 subtype avian influenza virus, there are still suspected cases, and it is easy to mix with Newcastle disease, which causes economic losses and brings a great challenge to the arrangement of epidemic prevention. Therefore, the combined immunization of avian influenza and Newcastle disease virus in order to prevent cross-infection and improve immune efficiency has become an important means of immunization in large-scale farms. In this study, XY-14 strain of H9 subtype avian influenza virus was isolated and identified from chicken farm of suspected low pathogenic avian influenza. After inactivation of the virus strain, combined immunization with Newcastle disease IV strain was carried out, and the change of antibody level in different immune methods was the focus of the study, and the speed and duration of antibody production by combined immunization of this strain and Newcastle disease virus were understood. This study mainly includes the following aspects: 1. In 2014, samples from Xinyang chicken farm were collected, inoculated with SPF chicken embryo and isolated and cultured. A strain of H9 subtype avian influenza virus was preliminarily screened by HA (hemagglutination test), HI (hemagglutination inhibition test) and RT-PCR method. The virus was purified by 50 times concentrated sucrose density gradient centrifugation after cloning and purification with limited dilution. The purified virus was amplified by Avian Influenza universal primer and H9 subtype avian influenza universal primer. Bright bands at approximately 229bp and 730bp positions, consistent with expected size. Specific primers were used to amplify the HA,NA,NP,M,NS gene fragment, and a bright band was found in 1740bpS1470bp 1565bp 1027bp and 890bp position respectively, which was consistent with the expected size, so the strain could be identified as H9 subtype. The virus strain was named A/Chicken/XinYang/XY-14/2014 (H9 Subtype), for short XY-14 strain). The titer of HA was 10log2H9 and HI H9 was 10log2.Then, the neutralization test was carried out with Avian Influenza virus H9 subtype serum. The collected allantoic fluid had no hemagglutination. 3. The virulence of the strain was determined and ICPI (1 day old chicken intracerebral inoculation pathogenicity index) ICPI=0.18; was done. And IVPI (chicken inoculation disease index) IVPI=0; The results showed that the XY-14 strain belonged to the attenuated strain. 4. After inactivating the XY-14 strain, the antibody titer reached a peak of 12 log2 at the 3rd week and the antibody level reached 10.33 log2 at the 22nd week. The results showed that the inactivated virus alone could make chickens produce antibody, and it could be provided with high titer of antibody for several weeks, and combined with Newcastle disease IV vaccine to immunize 0.5mL/ feathers, ND and AIV H9 antibodies were produced at the second week. The ND antibody reached a peak value of 11 log2 at week 3, and the antibody level of 6log2 remained at the end of 22 weeks. The peak level of AIV H9 antibody appeared in the 4th week, and up to the level of 11.33log2.ND 鈪，

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