鸡传染性支气管炎病毒(IBV)干扰新城疫病毒(NDV)增殖现象的研究

ＩＢＶ毒株Ｈ１２０、Ｈ５２、ＭＡ５对ＮＤＶ－ＬａＳｏｔａ的干扰实验证明ＩＢＶ特异性干扰ＮＤＶ增殖。１）采取不同顺序的同胚接种法：ＩＢＶ接种之后再接种ＮＤＶ;或ＮＤＶ接种之后再接种ＩＢＶ,及ＩＢＶ,ＮＤＶ同时接种,ＩＢＶ均干扰ＮＤＶ的增殖。２）ＮＤＶ接种３６小时之内,干扰现象最为明显,Ｈ１２０,Ｈ５２的干扰能力稍强于ＭＡ５。３）ＮＤＶ血清中和实验结果显示,不同顺序同胚接种ＮＤＶ、ＩＢＶ时,ＮＤＶ－ＬａＳｏｔａ不影响ＩＢＶ的增殖能力。同胚增殖ＩＢＶ,ＮＤＶ的关键是控制ＮＤＶ、ＩＢＶ的接毒量及选择合适的收毒时间。     

In ovo interference of embryo non-lethal avian infectious bronchitis viruses (IBV) with velogenic Newcastle disease virus and embryo adapted IBV

Avian infectious bronchitis virus (IBV) interfered with the lethal effects of velogenic Newcastle disease virus (NDV) and embryo adapted IBV in eggs previously inoculated with non-lethal IBV. Greater interference was noted in eggs superinfected with embryo adapted IBV than velogenic NDV. The interference could be eliminated by treating the initial IBV with homologous anti-IBV serum.     

鸡传染性支气管炎病毒对新城疫病毒的干扰作用观察

为观察鸡传染性支气管炎病毒（IBV）HN99株对新城疫病毒（NDV）增殖的干扰作用,该试验采用不同浓度的IBV标准株M41和地方株HN99与鸡新城疫病毒（NDV）分别按不同接种顺序同胚增殖,利用病毒血凝试验（HA）测定NDV的效价,观察IBV对NDV的干扰作用,从而为检测IBV地方株HN99提供方法,也为同胚增殖两种病毒提供一系列的数据参考。试验结果表明,鸡传染性支气管炎病毒地方株HN99对NDV的干扰作用与其浓度和接种顺序有关。     

鸡传染性支气管炎病毒与新城疫病毒增殖作用及免疫干扰的研究

试验旨在观察鸡传染性支气管炎病毒（infectious bronchitis virus,IBV）和鸡新城疫病毒（newcastle disease virus,NDV）之间的增殖干扰现象,分析在免疫过程中两种疫苗存在的免疫干扰,为确定疫苗的免疫程序提供依据。采用完全随机试验设计,选取10日龄鸡胚尿囊腔接种不同浓度的IBV、NDV以及不同浓度混合的IBV和NDV,收集尿囊液测定其血凝效价（HA）;用不同浓度的IBV、NDV以及不同浓度混合的IBV和NDV分别免疫BALB/c小鼠及SPF雏鸡,收集血清,间接酶联免疫吸附法（ELISA）和血凝抑制（HI）试验测定IBV和NDV的抗体效价及抑制效价。结果表明,同胚培养时,无论先接种IBV后接种NDV还是先接种NDV后接种IBV或IBV和NDV同时接种,IBV对NDV均有干扰作用,而NDV对IBV没有干扰作用;接种IBV和NDV的小鼠,其产生IBV和NDV的抗体效价均低于单独免疫组,免疫的次序及免疫间隔时间对IBV和NDV的抗体效价均有影响,IBV对NDV的免疫效果具有较大的干扰作用。不同针混合免疫方式能提高IBV和NDV的抗体效价;接种IBV和NDV的雏鸡,免疫次序及免疫相隔时间对IBV和NDV的抗体效价均有影响,IBV对NDV的免疫效果具有较大的干扰作用;雏鸡和小鼠免疫血清HI试验数据表明,免疫次序及免疫间隔时间对IBV和NDV抗体的产生均有影响,但对NDV抗体的产生影响更明显,随着免疫间隔时间的增加,IBV和NDV的抗体水平呈增加趋势,雏鸡比小鼠更能敏感地反映出IBV对NDV的免疫干扰作用。混合注射时IBV和NDV的抗体水平均降低,IBV对NDV的免疫效果有干扰作用。因此,在同胚培养、小鼠及雏鸡的免疫中,IBV对NDV有干扰作用,而NDV对IBV无干扰作用。混合接种时IBV和NDV抗体效价均下降,IBV对NDV的免疫效果有干扰作用。免疫次序及免疫间隔时间对IBV和NDV抗体的产生均有影响,但随着免疫间隔的增加,IBV和NDV的抗体水平呈增加趋势。     

Study on the Effect of Infectious Bronchitis Virus and Newcastle Disease Virus on Proliferation and Immune Interference

This experiment aimed to investigate the proliferation interference effects of the infectious bronchitis virus (IBV) and Newcastle disease virus (NDV),analyze the immune interference of two vaccines in the immune process,and provide a basis for determining vaccine immune program.This experiment was in a complete randomized design,10-day-old allantoic cavity of the chick embryo was inoculated with different concentrations of IBV and NDV and with a mixture of different concentrations of IBV and NDV,and then the allantoic fluid was collected for determination of the titer of hemagglutination (HA).Furthermore,BALB/c mice and SPF chicken were immunized with different concentrations of IBV and NDV and also with the mixed IBV and NDV with different concentrations,the blood of mice and chicken were collected,and antibody titer and inhibitory titer of IBV and NDV were determined by indirect enzyme linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test.Experimental results showed that in the homeomorphic cultivation research,whether IBV and NDV were inoculated in a different order or inoculated at the same time,the inoculation of IBV behaved interference to the NDV,while the inoculation of NDV had no interference effects to IBV;Mice were inoculated with IBV and NDV,IBV and NDV antibody titers were lower than single immunized group,immune procedures and immune intervals often had effect on the titer of IBV and NDV antibody,IBV had interference on the immune effect of NDV,different needle mixed immunization can improve the antibody titer of IBV and NDV.In the group of SPF chicken inoculated with IBV and NDV,the order of the inoculation and the immune interval had effect on the titer of IBV and NDV antibody,IBV had a interference on the immune effect of NDV;In the immune serum inhibitory test,the data showed that the IBV and NDV antibody production were affected by the immune order of the inoculation and the interval,the effect on the NDV antibody was more obvious,but with the increase of the immune interval,the antibody level of IBV and NDV showed an increasing trend,the chicken was more sensitive than the mice to reflect the immune interference of IBV on NDV.IBVand NDV antibodies titer was also reduced in the groups that was inoculated with the mixed IBV and NDV,IBV had a interference on the immune effect of NDV.In the homeomorphic cultivation research and animal research,IBV behaved interference to the NDV,while NDV had no interference effects to IBV.IBV and NDV antibody titers decreased when mixed immunization,IBV had a interference on the immune effect of NDV.The order of the inoculation and the immune interval had effect on the antibody titer of IBV and NDV,but with the increase of the immune interval,the antibody level of IBV and NDV showed an increasing trend.     

鸭新城疫病毒和流感病毒多重RT-PCR鉴别诊断技术的建立和应用

对同样以引起鸭产蛋下降为主要特征的鸭流感病毒（AIV）分离株和鸭新城疫病毒（NDV）YH99V分离株，通过设计特异性引物和优化体系反应条件，建立了一种早期快速鉴别诊断鸭AIV和鸭NDV的单项RT—PCR方法和多重RT—PCR方法，AIV的扩增片段大小为483bp，NDV的扩增片段为310bp，电泳快速，易于区分。敏感性试验表明，该方法比HA敏感100倍以上；对AIV、YH99V、IBV、IBDV及正常鸡胚尿囊液的混合液检测显示，多重RT—PCR法具有很强的特异性。对鸭病毒性产蛋下降征侯群送检病料的检测证明，该方法能有效鉴别AIv和／或NDV单独感染或混合感染，阳性检出率明显高于HA方法。     

鸡传染性支气管炎病毒CQ/01/2004株的分离与鉴定

2004年从重庆某肉用鸡场疑似鸡传染性支气管炎的病鸡中采集病料，按常规处理后接种9～10日龄鸡胚，通过鸡胚连续传代培养3代，并对该分离毒株的鸡胚致病性、血凝性和NDV的干扰特性进行检测．同时进行了动物回归试验。结果表明，该分离株具有IBV感染特征，可使鸡胚胚体出血、蜷缩、矮化；该分离毒株无直接血凝性，对NDV有明显的干扰作用；动物回归试验中有75％的感染鸡在10d内发病或死亡。剖检病死鸡可见肾苍白、肿胀，肾小管内充塞大量尿酸盐，支气管有出血点、有大量粘液。采用反转录．聚合酶链式反应（RT-PCR）技术对CQ／01／2004的纤突蛋白S1基因进行扩增、克隆和序列测定，结果表明该基因具有IBVSl基因的共有分子特征，将测序结果提交GenBank进行同源性检索，发现分离株CQ／01／2004和J株S1的同源性最高，核苷酸同源性为94％，氨基酸同源性为89．4％，与M41的核苷酸同源性为80．6％，氨基酸同源性为78．0％。试验结果表明，分离的病毒株CQ／01／2004为鸡传染性支气管炎病毒。     

Protection induced by infectious laryngotracheitis virus vaccines alone and combined with Newcastle disease virus and/or infectious bronchitis virus vaccines

Two types of live attenuated vaccines have been used worldwide for the control of infectious laryngotracheitis virus (ILTV): 1) chicken embryo origin (CEO) vaccines; and 2) tissue culture origin vaccines (TCO). However, the disease persists in spite of extensive use of vaccination, particularly in areas of intense broiler production. Among the factors that may influence the efficiency of ILTV live attenuated vaccines is a possible interference of Newcastle Disease virus (NDV) and infectious bronchitis virus (IBV) vaccines with the protection induced by ILTV vaccines. The protection induced by CEO and TCO vaccines was evaluated when administered at 14 days of age alone or in combination with the B1 type strain of NDV (B1) and/or the Arkansas (ARK) and Massachusetts (MASS) serotypes of IBV vaccines. Two weeks after vaccination (28 days of age), the chickens were challenged with a virulent ILTV field strain (63140 isolate, group V genotype). Protection was evaluated at 5 and 7 days postchallenge by scoring clinical signs and quantifying the challenge virus load in the trachea using real-time PCR (qPCR). In addition, the viral load of the vaccine viruses (ILTV, NDV, and IBV) was quantified 3 and 5 days postvaccination also using qPCR. The results of this study indicate that the NDV (B1) and IBV (ARK) vaccines and a multivalent vaccine constituted by NDV (B1) and IBV (ARK and MASS) did not interfere with the protection induced by the CEO ILTV vaccine. However, the NDV (BI) and the multivalent (B1/MASS/ARK) vaccines interfered with the protection induced by the TCO vaccine (P < 0.05). Either in combination or by themselves, the NDV and IBV vaccines decreased the tracheal replication of the TCO vaccine and the protection induced by this vaccine, since the ILTV-vaccinated and -challenged chickens displayed significantly more severe clinical signs and ILTV load (P < 0.05) than chickens vaccinated with the TCO vaccine alone. Although NDV and IBV challenges were not performed, the antibody responses elicited by NDV and/or the IBV vaccinations were significantly reduced (P < 0.05) when applied in combination with the CEO vaccine.     

鸡传染性支气管炎病毒地方流行株的分离与鉴定

从山西各地区疑似鸡传染性支气管炎(IB)的病料中,分离到5株鸡传染性支气管炎病毒(IBV)分离株,并对分离病毒进行了病毒形态观察、对鸡新城疫病毒(NDV)的干扰、鸡胚致病性试验、动物回归试验、血凝特性试验、病毒理化特性测定等生物特性鉴定及IBV N基因特异性片段的检测.电镜观察,可见直径为60～120 am,有囊膜及纤突呈冠状排列的病毒粒子;对NDV有明显的干扰作用;分离株的传代物均有明显的致鸡胚矮小化作用;动物回归感染死亡鸡肾脏病变明显,表现肾脏肿大、花斑肾现象,输尿管内充塞大量尿酸盐;无直接血凝性,经1%胰酶处理后可凝集鸡红细胞;分离株对乙醚和氯仿敏感;采用反转录-聚合酶链式反应(RT-PCR)对分离毒株进行扩增,结果均扩增出特异N基因核酸片段.     

巢式PCR快速鉴定鸡传染性支气管炎病毒的研究

鸡传染性支气管炎病毒（ＩＢＶ）经鸡胚增殖后,直接用尿囊液提取ＲＮＡ后反转录成ｃＤＮＡ,用ＩＢＶ基因３’端的ＵＴＲ１－／ＵＴＲ２＋和ＵＴＲ３－／ＵＴＲ４＋两对引物进行巢式ＰＣＲ,所检测的４个ＩＢＶ标准参考株和１６个ＩＢＶ野毒株均得到了预期的１７４ｂｐ大小的片段,而鸡新城疫病毒（ＮＤＶ）,鸡传染性法氏囊病毒（ＩＢＤＶ）及正常鸡胚尿囊液经同样处理没有可见片段出现。本试验不需纯化册毒只需０５ｍｌ病毒尿囊液即可在２４小时内得到准确的试验结果。这表明与其它ＩＢＶ鉴定方法相比,该法具有快速、灵敏、特异的优点。     

The immune response of the chick following viral vaccinations and immunization with sheep red blood cells

The effects of viral vaccinations and immunization with sheep red blood cells (SRBC) on the humoral response of pullets were investigated. Pullets were vaccinated with Marek's disease virus, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and infectious bursal disease virus at appropriate ages used in commercial practice. At seven weeks, the pullets were intramuscularly immunized with SRBC. NDV and IBV antibodies were detected by hemagglutination-inhibition tests. Hemagglutination (HA) titers were established against SRBC. IBV antibody titers were not affected by vaccination or by immunization with SRBC. NDV antibody titers were significantly increased by vaccination and by immunization with SRBC. The SRBC agglutinin response was also positively affected by vaccination. The HA titer increase consisted of a rise in 2-mercaptoethanol (2-ME)-sensitive antibodies and a fall in 2-ME-resistant antibodies.     

陕西省鸡肾型传染性支气管炎病毒的分离鉴定

从临床表现呼吸道传染病的20个发病鸡群中先后分离出18株病毒,通过鸡胚感染、血清学试验、分子生物学试验、动物回归试验证明其中13株是冠状病毒科的鸡传染性支气管炎病毒,3株为新城疫病毒,2株为H9禽流感病毒。该13株中的BJ2、FF2、YL2、B01、G5、YX、WH、WG分离毒具有很强的嗜肾性和致病力,这8个陕西流行株的血清型与W118毒株相同,而与IBVGray株、W93株有一定的差异。结果表明,陕西地区主要流行鸡肾型传染性支气管炎,且流行株具有强致病力。     

鸡新城疫病毒与传染性支气管炎病毒同胚培养的试验研究

将鸡新城疫病毒（NDV）和鸡传染性支气管炎病毒（IBV）按一定的稀释比例等量混合接种9~10日龄SPF鸡胚,让其在同一鸡胚中增殖（简称同胚培养）。试验结果表明,用5倍稀释的ND-Lasota病毒液与 5000倍稀释的IBH₁₂₀病毒液等体积混合后尿囊腔接种鸡胚,能在同一鸡胚中正常增殖,接种后96 h收毒,用红细胞凝集试验（HA）测得NDV、IBV两种病毒的血凝价分别为11 log2和12 log2,不低于各自病毒单独增殖的HA滴度,病毒的增殖基本趋于平衡。同时也证实了在合适的培养条件下,IBV对NDV的复制不会产生干扰。     

Characterization of infectious bronchitis virus using monoclonal antibodies

Three monoclonal antibodies (MABs) reactive against two structural proteins--the nucleoprotein (NP) or the surface (S) protein--of avian infectious bronchitis virus (IBV) were produced and characterized. The MABs did not neutralize virus infectivity or inhibit hemagglutination. Their reactivity patterns with the homologous strain and eight heterologous strains of IBV were determined using the indirect immunoperoxidase test, the indirect immunofluorescent test, transfer-immunoblotting of separated proteins, and a dot-immunoblotting assay (DIA). Two MABs, NP- or S-protein-specific, reacted with all nine strains; one (NP-specific) reacted with only two strains. The two MABs reacting with all nine strains of IBV also detected 18 IBV field isolates of unknown serotype in the DIA. The MAB detecting only two strains did not react in the DIA. The diagnostic application of these MABs appears promising.     

Evaluation of different methods of inactivation of Newcastle disease virus and avian influenza virus in egg fluids and serum

Viruses conveyed in shipments of eggs, viral diagnostic reagents, or avian serum samples are a potential hazard for susceptible poultry. Different methods of treatment of those materials to eliminate the hazard of virulent and avirulent strains of Newcastle disease virus (NDV) or avian influenza virus (AIV) were evaluated. The NDV strains tested were more thermostable than the AIV strains. The results suggest that standard pasteurization methods would not reliably inactivate the concentrations of NDV used. beta-Propiolactone (BPL) (greater than or equal to 0.025%) inactivated NDV or AIV in allantoic fluid, but higher concentrations were needed to inactivate virus diluted in serum. Hemagglutination (HA) of NDV and AIV and hemolysis (HL) activity of NDV were reduced or eliminated by 0.4% BPL. Formalin (greater than or equal to 0.04%) inactivated either virus but adversely affected HA and HL activity. NDV or AIV was inactivated by binary ethylenimine (BEI) (0.01 M) with no adverse effect on HA or HL. Heat (56 C) or BEI (0.01 M) had no apparent effect on hemagglutination-inhibition (HI) titers of NDV and AIV antisera, the effect of formalin (0.1%) was variable, and BPL (greater than or equal to 0.25%) depressed the HI titers of both antisera. The optimum method should achieve virus inactivation without harming the treated material.     

Use of a constant-virus diluting-serum microneutralization technique for the detection and quantification of infectious bronchitis virus antibodies

A constant-virus diluting-serum microneutralization test (CVMNT) for avian infectious bronchits virus (IBV) was evaluated for both reliability and repeatability. The virus used in the assay was a chick kidney (CK) cell-adapted strain, the Beaudette strain (IBV 42). Sera tested were from 24-week-old broiler-breeder chickens that had been vaccinated 3 times from a combination vaccine of Newcastle disease virus (NDV) and IBV. Test results were not repeatable or comparable when the same sera were tested on different days, but test results were repeatable and comparable when the sera were tested on the same day. Differences in virus titer at the different times that tests were performed appeared to cause the variation in test results. A comparison was made between the CVMNT and a constant-serum diluting-virus microneutralization test (CSMNT). The CVMNT was able to detect differences in flock antibody titers that the CSMNT could not.     

一株鸽新城疫病毒的分离鉴定

对广东地区疑似发生鸽新城疫感染的鸽病料进行病毒分离,通过血凝试验(HA)、中和试验、F基因扩增及序列测定.结果分离到1株血凝效价为4log2,且能被NDV阳性血清中和的病毒;用针对NDV F基因设计的特异性鉴定引物对该分离株进行PCR扩增,可扩增出相应的目的片段;测序及Blast分析表 明其与山东分离株chicken/...