Comparison of serological tests for antibodies against Newcastle disease virus and infectious bronchitis virus using ImmunoComb solid-phase immunoassay, a commercial enzyme-linked immunosorbent assay, and the hemagglutination-inhibition assay

COMBSCORES determined using the ImmunoComb solid-phase immunoassay were compared with hemagglutination-inhibition (HI) titers specific for Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) and with mean enzyme-linked immunosorbent assay (ELISA) titers determined using Agritech Systems, Inc., ELISA. COMBSCORES for NDV and IBV increased proportionately in a stepwise manner as HI titers increased. The ImmunoComb solid-phase immunoassay was ablt to produce endpoint titers on sera with NDV-HI titers of 0 through 320 and IBV-HI titers of 0 through 1024 without reaching the maximum S-value. The ImmunoComb showed good correlation with the HI assay and the Agritech ELISA and should prove to be a useful tool for serological profiling, either alone or in conjunction with the HI test or commercial ELISA.     

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.     

Immune response to oil-emulsion vaccines with single or mixed antigens of Newcastle disease, avian influenza, and infectious bronchitis

Inactivated Newcastle disease virus (NDV), avian influenza virus (AIV), and infectious bronchitis virus (IBV) antigens were evaluated for immunological efficacy in monovalent and polyvalent vaccines. Vaccinated broilers were bled for hemagglutination-inhibition (HI) tests at 1- or 2-week intervals. Half of the chickens were challenged with the Largo isolate of velogenic viscerotropic (VV) NDV at 8 weeks post-vaccination, and the remainder were challenged with the Massachusetts 41 strain IBV at 9 weeks post-vaccination. Newcastle disease HI titers were reduced significantly (P less than 0.05) from those of monovalent control vaccine groups when IBV antigen was emulsified in mixtures with low (1-3x) concentrated NDV or NDV and AIV antigens. Avian influenza HI titers were significantly (P less than 0.05) lower than those of the control monovalent groups when highly concentrated NDV was part of the polyvalent vaccine. Infectious bronchitis HI titers were higher than those of control monovalent groups in 13 of 15 vaccine groups when IBV antigen was in polyvalent formulations. VV NDV challenge killed all non-NDV vaccinates and induced increased HI titers in NDV vaccinates but no morbidity or mortality. Sixty of 80 IBV vaccinates experienced a fourfold or greater HI titer increase following challenge. All non-IBV vaccinates seroconverted at 1 week post-challenge.     

Comparison of egg-yolk and serum antibody titers to four avian viruses by enzyme-linked immunosorbent assay using paired field samples

Eggs and blood were collected from 11 hens in each of nine broiler-breeder flocks in Quebec. Serum and egg-yolk extracts were assayed for antibody titers to infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and reovirus (RV) by a commercial enzyme-linked immunosorbent assay (ELISA) kit. Comparison was made between egg-yolk and serum antibody titers by a regression analysis. A high correlation was observed between serum and yolk antibody titers to all the viruses tested (r = 0.9 for IBDV, 0.84 for IBV, 0.84 for NDV, and 0.91 for RV). Antibody monitoring of commercial breeder flocks using egg yolk instead of serum with commercial ELISA plates is thus feasible and is recommended.     

Comparison of two commercial enzyme-linked immunosorbent assays and conventional methods for avian serology

Sera tested for hemagglutination-inhibition (HI) activity against Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) and virus-neutralizing (VN) activity against infectious bursal disease virus (IBDV) and viral arthritis (VA) virus were collected from a wide variety of accessions into the Diagnostic Services Laboratory, Poultry Disease Research Center, University of Georgia. The sera were then segregated according to HI or VN titer to NDV, IBV, IBDV, or VA virus and stored frozen at -20 C until tested by two commercial enzyme-linked immunosorbent assays (ELISAs). There was good correlation of mean Flockchek ELISA titers or EIA Systems sample-to-positive (S/P) ratios with specific HI or VN titers. Flockchek ELISA profile group 3 and EIA Systems mean S/P ratio of 1.12 corresponded to what were considered in our lab to be minimum protective titers for each antigen against virulent challenge in our area.     

朗德鹅禽流感病毒的分离与鉴定

用禽流感病毒ELISA试剂盒对某朗德鹅养殖场的病鹅气管粘液进行了检测，发现5份粘液样本均呈禽流感阳性；随后取相应气管组织材料接种于9～11日龄鸡胚分离病毒．发现尿囊液能使鸡红细胞发生凝集，用禽流感病毒H5、H7、H9标准阳性血清和新城疫病毒、传染性支气管炎病毒、传染性喉气管炎病毒、传染性法氏囊炎病毒抗血清作HI试验，结果禽流感病毒H5亚型抗血清的血凝抑制滴度达到2^7，而禽流感病毒H7、H9亚型及其他病毒抗血清无血凝抑制滴度，说明从朗德鹅分离到的病毒为H5亚型禽流感病毒。     

Genetic variation in resistance of turkeys to experimental infection with Newcastle disease virus.

Seven hundred eighty male and female turkeys representing four genetic lines were challenged in four experiments with the Texas GB strain of Newcastle disease virus (NDV). The lines of turkeys included two randombred control lines (RBC1 and RBC2), a subline (E) of RBC1 selected for increased egg production, and a subline (F) of RBC2 selected for increased 16-week body weight. Mortality in turkeys of subline F (32.5%) was significantly higher than that in turkeys of line RBC2 (15.8%), subline E (17.5%), and line RBC1 (18.4%). At the end of each experiment, surviving birds were tested for antibody to NDV using the enzyme-linked immunosorbent assay (ELISA) and hemagglutination-inhibition (HI) test. Turkeys of subline E and line RBC1 had significantly lower ELISA antibody titers than those of subline F and line RBC2. Subline F had the highest HI antibody titers, followed in decreasing order by lines RBC2 and RBC1 and subline E. No apparent correlation was found between antibody response and mortality after NDV challenge.     

Infectious bronchitis virus serotypes in poultry flocks in Jordan

Infectious bronchitis virus (IBV) causes respiratory disease in chickens all over the world. IBV has many serotypes that do not confer cross protection against each other. Hemagglutination inhibition (HI) test has been used to determine the serotypes of IBV as a substitute to the more laborious virus neutralization test and the more sophisticated restriction endonuclease digestion or sequencing of the S1 gene. In Jordan, no previous studies have been carried out to determine the involvement of IBV in respiratory disease in chickens, or the serotypes of IBV that possibly exist. In this study, serum from different chicken flocks (n = 20) that suffered from respiratory disease were tested for IBV antibodies using commercial IBV antibody ELISA at time of the initial signs of the respiratory disease and repeated on serum samples from the same flocks 10–14 days later. ELISA titer for IBV increased in 14 out of 20 flocks (70%) after 10–14 days of the initial signs of the respiratory disease and this indicates a recent exposure to IBV. The second serum samples from these 14 flocks were further examined against a panel of five IBV antigens (Ark, Conn, DE-072, JMK, and Mass) by HI test to determine the serotype(s) of IBV they have been exposed to. The HI test results indicated that the exposure of some of these flocks were to Ark, DE-072, and Mass like serotypes. However, the HI titers against the antigens used in this study were relatively similar in 10 out of the 14 flocks (71%) and the serotype of IBV that these flocks were exposed to could not be determined and the possible causes of this are discussed.     

Serosurvey of five viruses in chickens on smallholdings in Bangladesh

A serologic survey was undertaken in chickens in smallholdings in Bangladesh for avian influenza A virus (AIV), egg drop syndrome '76 virus (EDS'76V), infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and reovirus (RV) in three phases: January 2002-May 2003, September 2003-August 2004, and August 2005-March 2006. Four hundred thirty-six sera collected in the 2nd phase, 295 in the first phase, 755 in the 1st plus 2nd phases and 295 in the 1st phase were investigated for AIV, EDS'76V, IBV and RV, respectively, using enzyme linked immunosorbent assays. All 854 sera collected in the three phases were screened for NDV using hemagglutination inhibition test. In chickens 20% were seropositive to AIV, 3% to EDS'76V, 74% to IBV, 88% to NDV, and 47% to RV. The seroprevalence in flocks was 23% to AIV, 6% to EDS'76V, 79% to IBV, 89% to NDV and 56% to RV. Twenty-five percent chickens had > or = 10log(2)HI titers to NDV.     

Rapid serological profiling by enzyme-linked immunosorbent assay. I. Measurement of antibody activity titer against Newcastle disease virus in a single serum dilution

An enzyme-linked immunosorbent assay (ELISA) was developed to measure specific antibody activity in sera of chickens exposed to Newcastle disease virus (NDV). A near-linear relationship existed between the log of the corrected absorbance of antisera at a single working dilution and the corresponding observed serum titers as determined by a standard serial-dilution method. Regression analysis was used to construct a standard curve and extract an equation from this relationship. The equation was used to convert corrected absorbance readings of the single working dilution directly into predicted ELISA antibody activity titers. In a comparative study, a correlation (P less than 0.01) was found between ELISA and hemagglutination-inhibition (HI) antibody titers to NDV. ELISA titers were as much as 160 times greater than the HI titers. ELISA was also able to detect much lower levels of antibody activity than the HI test.     

鸵鸟新城疫病毒野毒株的分离及其生物学特性

用鸡胚从疑似鸵鸟新城疫的送检病料中分离到 2株病毒。 2株病毒均能凝集鸡红细胞 ,且能被抗新城疫病毒 ( NDV)阳性血清抑制 ;用抗 NDV单抗 PEG夹心 ELISA测定 2株分离毒均为阳性。对其中 1株作进一步生物学特性鉴定 ,按照国际上规定的 NDV毒力判定标准测定的该毒株最低致死量致死鸡胚的平均死亡时间 ( MDT)为 54h,1日龄雏鸡脑内接种致病指数 ( ICPI)为 1 .88,6周龄雏鸡静脉接种致病指数 ( IVPI)为2 .75。结果表明 ,本分离株为鸵鸟新城疫病毒强毒     

免疫层析试纸条(ICS)检测禽流感抗体

以纯化的禽流感病毒（avian influenza virus,AIV）核蛋白作为捕捉抗原，金标兔抗鸡抗体作为金标二抗，建立检测禽流感血清抗体的胶体金免疫层析试纸条（immunochromatographic strip, ICS）。对标准阳性血清不同滴度进行检测，ICS试验的敏感性高于琼脂扩散试验（AGP）。特异性试验证实，禽流感的ICS试验与新城疫（ND）、传染性支气管炎（IBV）、鸡传染性法氏囊病（IBD）及减蛋综合症（EDS-76）均无交叉反应。同时，用ICS试验和HI试验检测105份鸡血清，两者具有较好的符合性。结果认为：禽流感ICS具有快速简便、特异、灵敏等优点。     

鸡新城疫、减蛋综合症、传染性支气管炎三联油佐剂灭活苗的研究Ⅱ、不同单位病毒配比的三联苗对鸡免疫抗体产生的影响

分别用三个滴度的ＮＤＶ、ＥＤＳ_７６Ｖ、ＩＢＶ作正交配比，制成八组不同配比的三联油佐剂灭活苗，鸡免疫后每月采血分离血清，应用ＨＩ监测其抗体，结果表明，三种病毒适当配比，不产生干扰，疫苗的免疫效果与抗原量有关。本疫苗与Ｉｎｔeｒｖｃｔ公司生产的ＮＤ－ＩＢ－ＩＢＤ三联油佐剂灭活苗作比较试验，免疫鸡所产生的ＮＤ、ＩＢ抗体无显著性差异。应用含抗原量最低口比的疫苗，对不明原因的有产蛋率下降疫病感染史的鸡群作免疫接种，冬季该场蛋鸡群普遍发生产蛋率明显下降的疫病，用本苗免疫的鸡群产蛋享未受影响。     

中药制剂对禽流感病毒H9N2、新城疫病毒、传染性支气管炎病毒和鸡毒支原体的攻毒保护试验

采用以野菊花、穿心莲、柴胡等10味中药提取的中药制剂,连续给22日龄SPF鸡口服5 d后,以禽流感病毒H9N2、新城疫病毒F4、传染性支气管炎病毒强毒株M41和支原体S6强毒分别攻击,观察SPF鸡临床症状、发病率、病理学变化和分离病毒。结果表明,此中药制剂75 mg/只口服组,新城疫病毒、禽流感病毒和传染性支气管炎病毒攻击后发病率分别为12.5%,0%,10%;37.5 mg/只剂量口服组发病率分别为50%,10%,10%,且低剂量组病毒分离阳性。攻击支原体后,以上两个剂量组SPF鸡病变指数分别3.5和5.5,研究证实本中药制剂可以控制以上4种病原所致的家禽呼吸道病的发生。     

The effect of dietary lysine deficiency on the immune response to Newcastle disease vaccination in chickens

The effect of lysine deficiency on chicken immune function was evaluated using broiler chickens fed a diet with lysine at 67% of the control diet (1.24% lysine). The evaluation of humoral immune function was conducted by measuring the antibody production to a live Newcastle disease virus (NDV) vaccination using the hemagglutination inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA). The cellular immune function was evaluated through the use of cutaneous basophil hypersensitivity test. The antibody response to NDV vaccination was reduced in broiler chickens fed a lysine-deficient diet when measured by ELISA but not when measured by HI. The cell-mediated immune response was also reduced by lysine deficiency.     

Development and application of a multiplex polymerase chain reaction for avian respiratory agents

A multiplex polymerase chain reaction (PCR) was developed and optimized to simultaneously detect 6 avian respiratory pathogens. Six sets of specific oligonucleotide primers for infectious bronchitis virus (IBV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), Newcastle disease virus (NDV), Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS) were used respectively in the test. With the use of agarose gel electrophoresis for detection of the PCR-amplified DNA products, the sensitivity of detection was between 10 pg for IBV, AIV, MG, and ILTV and 100 pg for NDV and MS after 35 cycles of PCR. Similar sensitivity of these primers was achieved with chickens experimentally infected with respiratory pathogens. In experimental infections, the multiplex PCR was able to detect all the infected chickens in each group at I and 2 wk postinfection as compared with serologic tests at 2 wk postinfection that confirmed the presence of specific antibodies. The multiplex PCR was also able to detect and differentiate coinfections with two or more pathogens. No specific DNA amplification for respiratory avian pathogens was observed among noninoculated birds kept separately as a negative control group.     

A Serological Survey for Avian Infectious Bronchitis Virus and Newcastle Disease Virus Antibodies in Backyard (Free-range) Village Chickens in Mexico

The commercial flocks in Yucatan, Mexico are free of Newcastle disease virus (NDV) in its velogenic viscerotropic form, but little is known about the disease status of backyard poultry. A seroprevalence survey in 30 villages using haemagglutination inhibition (HI) tests for infectious bronchitis virus (IBV) and NDV antibodies was carried out from December 1997 to June 1998. The seroprevalences were 56.5% (95% CI 50–63%) for IBV and 2.2% (95% CI 0.5–3.8%) for NDV. All the villages had chickens that were positive for antibodies to IBV and nine of the villages had chickens that were positive for antibodies to NDV. This suggests that IBV may be responsible for a large proportion of the respiratory disease observed in backyard chickens in Yucatan. The implications of these findings are discussed, including the highly susceptible status of the backyard chickens in Yucatan to NDV and the possibility of this virus being one cause of the syndrome known as mortandad by the local people.     

应用多重聚合酶链反应同时检测鉴别鸡新需疫病毒,鸡传染性支气管炎病毒,鸡

本文建立了一种同时检测ＤＮＶ、ＩＢＶ、和ＭＧ４种病原体的多重ＰＣＲ技术。根据ＮＤＶ、ＩＢＶ、ＩＬＴＶ和ＭＧ的基因文库,设计了４对分别与ＮＤＶ、ＩＢＶ、ＩＬＴＶ和ＭＧ某段基因序列互补的引物,用这４对引物对同一样品中的ＮＤＶ、ＩＢＶ、ＩＬＴＶ、ＭＧＲＶＡ和ＤＮＡ模板进行多重ＰＣＲ扩增,结果均同时得到了４条特异性大小与实验设计相符的３１０ｂｐ（ＮＤＶ）、１７２０ｂｐ（ＩＢＶ）、６４７ｂｐ（ＩＬＴＶ）和７３２ｂｐ（ＭＧ）多重ＰＣＲ扩增带,而对其他６种禽病病原的ＰＣＲ扩增结果均为阴性;敏感性测定结果表明,该多重ＰＣＲ技术难检出１０ｐｇ的ＩＢＶ、１ｂｇ的ＮＤＶ ＲＮＡ模板和ＩＬＴＶ、１ｐｇ的ＭＧ ＤＮＡ模板。