H9亚型禽流感病毒XY-14株和鸡新城疫病毒La Sota株联合免疫的研究
发布时间：2018-12-10 16:35
【摘要】：H9亚型禽流感是目前危害我国养禽业的主要AIV亚型,以引起病鸡体温升高和呼吸道病变,成年鸡产蛋量下降,畸形蛋增多为主要特征。近年来,有研究表明在进行H9亚型禽流感病毒免疫后,仍不断有疑似病例发生,且极易与新城疫发生混合感染,造成经济损失的同时给防疫安排工作带来了很大的挑战。因此,将禽流感和新城疫病毒进行联合免疫,以防止交叉感染,提高免疫效率,成为如今规模化养殖场免疫的重要手段。本研究从疑似低致病性禽流感发病鸡场采集病料,分离鉴定出H9亚型禽流感病毒株XY-14株。将此毒株灭活后与新城疫Ⅳ系进行联合免疫,以不同免疫方式产生抗体水平的变化为研究重点,了解此毒株与新城疫病毒联合免疫产生抗体的快慢与持续期。本研究主要包括以下几个方面:1、2014年从信阳鸡场采集疑似低致病性禽流感发病的病料,接种SPF鸡胚,分离培养病毒。通过HA(血凝试验)、HI(血凝抑制试验)和RT-PCR方法初步筛到1株H9亚型禽流感病毒。将病毒进行终点有限稀释克隆纯化后50倍浓缩蔗糖密度梯度离心提纯,取纯化好的病毒用禽流感通用引物和H9亚型禽流感通用引物进行扩增,在大约229bp和730bp位置得到明亮的条带,与预期大小一致。使用特异性引物扩增HA、NA、NP、M、NS基因片段,分别在1740bp、1470bp、1565bp、1027bp和890bp位置出现明亮的条带,与预期大小一致,因此可确定该毒株为H9亚型。将此毒株命名为A/Chicken/XinYang/XY-14/2014(H9 Subtype),简称为XY-14株。2、XY-14株的HA效价为10log2,HI H9效价为10log2,然后用禽流感病毒H9亚型血清进行中和试验,收集的尿囊液没有血凝性。3、对该毒株进行毒力测定,做ICPI(1日龄鸡脑内接种致病指数统计)ICPI=0.18;和IVPI(鸡静脉接种致病指数)IVPI=0;说明XY-14株属于弱毒株。4、将XY-14株病毒进行灭活处理,单独免疫鸡,第3周抗体滴度达到峰值12log2,持续直到第22周抗体水平仍达到10.33log2。结果表明该灭活毒单独免疫,能够使鸡产生抗体,且能以很高的抗体滴度提供持续数周;与新城疫Ⅳ系苗联合免疫0.5mL/羽,第2周便产生ND和AIV H9抗体;ND抗体在第3周达到峰值11log2,持续到22周仍有6log2的抗体水平;AIV H9抗体水平峰值于第4周出现,高达11.33log2。NDⅣ系滴鼻点眼同时再以0.3mL/羽进行联合毒株免疫时,不但能同时产生抗两种病毒的抗体,还能显著提高ND抗体滴度,但不对抗H9 HI水平产生影响,说明联合免疫的两种病毒之间互不干扰;联合免疫0.3mL/羽,同时NDⅣ系滴鼻点眼,在本实验中为较适免疫方式。
[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，
本文编号：2370884
[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，
本文编号：2370884
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