Development and evaluation of an ELISA for the detection of antibody to infectious laryngotracheitis virus in chickens

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to infectious laryngotracheitis (ILT) virus in chickens was developed and compared with the serum-neutralization assay. The ELISA routinely yielded 16-to-32-fold higher titers than the serum-neutralization test. To overcome the requirement for large amounts of purified viral antigen, the microtiter trays were initially coated with an antibody prepared against purified ILT virus. A relatively crude viral preparation could then be used to coat the trays. Sera from specific-pathogen-free chickens less than 12 weeks of age did not show nonspecific binding, although 2.7% of all sera from chickens between 13 and 64 weeks of age had nonspecific activity. The majority of nonspecific reactors came from one highly inbred flock of specific-pathogen-free chickens. A number of modifications of ELISA procedures reported to reduce the nonspecific binding of chicken sera were investigated. Treatment of the serum or the plate and changes in the composition of the diluent did not increase the relative sensitivity of the anti-ILT assay.     

Sensitivity and specificity of an enzyme-linked immunosorbent assay for the detection of infectious bovine rhinotracheitis viral antibody in cattle.

An enzyme-linked immunosorbent assay was developed to detect bovine serum antibody to infectious bovine rhinotracheitis virus. The specificity of this assay in 304 bovine sera, collected from an infectious bovine rhinotracheitis virus-free herd, was 100%; in sera from 62 cattle inoculated with an intranasal vaccine, its diagnostic sensitivity was 27.4% at one month and 100% at six months, postvaccination. In 303 bovine sera with standard serum neutralizing antibody titers of greater than or equal to 1:2 it showed 100% sensitivity; and in 463 random diagnostic samples, comparative tests indicated that enzyme-linked immunosorbent assay detected more seropositive animals (61.6%) than the standard serum neutralizing test (49.9%). The enzyme-linked immunosorbent assay method was considered to be technically superior as a routine diagnostic test for the detection of infectious bovine rhinotracheitis viral antibody in bovine sera.     

鸡传染性喉气管炎病毒TaqMan real-time PCR检测方法的建立

为建立鸡传染性喉气管炎病毒(ILTV)TaqMan Real-time PCR检测方法,本研究根据GenBank中登录的ILTV gB基因序列设计了2对引物与一条特异性TaqMan探针,通过对反应体系和反应条件的优化,特异性、敏感性以及重复性试验,证明该方法在核酸含量108拷贝/μL～101拷贝/μL范围内具有良好的线性关系;能够检测初始模板中10-3EID50的病毒核酸及16拷贝的标准品;与其它相关的鸡源病毒均无交叉反应,并且批内、批间变异系数均小于2%,具有良好的重复性。该检测方法的建立为ILTV的临床检测和定量分析提供了一种快速、准确的技术手段。     

Sensitivity and specificity of an enzyme-linked immunosorbent assay for the detection of bovine viral diarrhea virus antibody in cattle.

A reliable bovine viral diarrhea (BVD) viral antigen was prepared from BVD virus grown on Madin Darby bovine kidney (MDBK) cells by solubilizing the virus with detergent MEGA-10 (decanoyl-N-methylglucamide) followed by removal of hydrophobic proteins with Triton X-100 treatment. By these treatments, problems of high background associated with BVD viral antigen in the enzyme-linked immunosorbent assay (ELISA) were eliminated. With this new antigen, an ELISA was adapted to detect bovine serum antibody against BVD virus. The diagnostic specificity of the assay in 403 bovine sera collected from a BVD virus-free herd was 100%; in 296 bovine sera with serum neutralizing antibody titers of greater than or equal to 1:2, 289 sera were ELISA positive (relative sensitivity of 97.6%), two sera gave false negative reactions (0.7%) and five sera gave suspicious reactions (1.7%). These interpretations were based on positive/negative (P/N) ratio readings, i.e. a P/N ratio of less than 1.50, 1.50-1.99 and greater than or equal to 2.00 were interpreted as negative, suspicious and positive reactions, respectively. The ELISA results gave excellent agreement with serum neutralization in detecting both seropositive and seronegative animals (Kappa = 0.994). The ELISA assay was considered to be technically superior to the serum neutralization test for the routine detection of BVD viral antibody in bovine sera.     

Labeled avidin-biotin enzyme-linked immunosorbent assay for detecting antibody to infectious laryngotracheitis virus in chickens

A labeled avidin-biotin enzyme-linked immunosorbent assay (LAB-ELISA) for detecting antibody to infectious laryngotracheitis (ILT) virus in chicken sera was developed and compared with ordinary ELISA. Purified ILT virus, biotin-labeled anti-chicken IgG rabbit IgG conjugate, and horseradish-peroxidase-labeled avidin were used in the LAB-ELISA. When sera from farm chickens were tested by serum neutralization (SN) and two kinds of ELISA, the correlation rate between SN and LAB-ELISA was 50/50 (100%), and that between SN and ordinary ELISA was 39/50 (78%). In LAB-ELISA, all of the sera that were antibody-negative by SN had low absorbance (A) values (below 0.05), and the A values were closely correlated with the SN indexes. In ordinary ELISA, however, the sera antibody-negative by SN had various A values ranging from 0.06 to 0.32. LAB-ELISA had much lower nonspecific reactions than ordinary ELISA against sera from ILT-negative chickens, even when chickens were 30 weeks old. ILT antibody production after ILT vaccination could be detected by LAB-ELISA. A values peaked 5 weeks postinoculation and were maintained for 17 weeks.     

Identification of the thymidine kinase gene of infectious laryngotracheitis virus.

An an initial step in the development of a recombinant poultry infectious laryngotracheitis virus (ILTV) vaccine, we report on the identification, cloning, and sequencing of a thymidine kinase (tk) gene from a virulent U.S. field isolate of ILTV. Degenerate oligonucleotide primers for the consensus nucleotide (ATP) binding site and the nucleoside (thymidine) binding site of other herpesvirus tk genes were used in the polymerase chain reaction (PCR) to amplify a fragment of ILTV DNA. The 344-base-pair (bp) amplified fragment was cloned into plasmid pKSII and used in Southern hybridizations to locate the ILTV tk gene on a 2.4-kb HindIII fragment. Upon cloning and sequencing this fragment, a 1089-bp open reading frame was identified, which is predicted to encode a protein demonstrating 27.9% amino acid homology to the herpes simplex virus type 1 (HSV-1) thymidine kinase protein. Analysis of the sequence revealed one region of difference from that reported for the Thorne strain of ILTV. In addition, the portion of the TK protein corresponding to the nucleotide binding domain is highly conserved among the avian herpesviruses.     

Reproducibility of a virus-neutralization test for infectious laryngotracheitis virus

A virus-neutralization test for infectious laryngotracheitis virus was performed in microtiter plates using standard techniques. To assess the reproducibility of the test, 11 sera were each titrated repeatedly once a week for 8 weeks, and the results were compared to a standard. The standard used for reproducibility was that the 95% logarithmic confidence intervals of the mean calculated from three titrations of the same serum had to be smaller than the logarithmic distance "within" two microtiter plate wells. For the virus neutralization test to give reproducible results, such confidence intervals had to fall "within" two wells at least 75% of the time. Over the 8 weeks, percent reproducibility varied from 43.5% to 81.5%. The infectious laryngotracheitis virus-neutralization test did not meet our defined standard of reproducibility with positive antisera. Results with negative control sera were reproducible, however. Percent reproducibility varied from 31.8% to 93.8% for different sera tested, but it was not related to the titer of the sera.     

Molecular biology of avian infectious laryngotracheitis virus

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes an economically important chicken disease, which results in delayed growth, reduced egg production, and also frequently in death of the animals. After acute infection of the upper respiratory tract, the virus can establish latency in the central nervous system, and subsequent reactivations can lead to infection of naive chickens. For prevention of ILT, conventionally attenuated live vaccines are available. However, these vaccine strains are genetically not characterized, and reversions to a virulent phenotype occur. Although molecular analyses of ILTV are hampered by the lack of an optimal cell culture system, the complete nucleotide sequence of the ILTV genome has recently been elucidated, and several ILTV recombinants lacking nonessential, but virulence determining genes have been constructed. Animal trials indicated that genetically engineered stable gene deletion mutants are safe alternatives to the current vaccine strains. Furthermore, since live ILTV vaccines are suitable for fast and inexpensive mass administration, they are promising as vectors for immunogenic proteins of other chicken pathogens. Thus, immunization with ILTV recombinants expressing avian influenza virus hemagglutinin was shown to protect chickens against ILT and fowl plague. Using monospecific antisera and monoclonal antibodies several virion proteins of ILTV have been identified and characterized. Since they include immunogenic envelope glycoproteins, these results can contribute to the improvement of virus diagnostics, and to the development of marker vaccines.     

应用间接荧光抗体技术检测兔波氏杆菌

以兔波氏杆菌为免疫原,强化免疫家兔,制备兔抗血清为第一抗体;以标准的羊抗兔IgG-FITC荧光抗体为第二抗体,建立了检验兔败血波氏杆菌的间接荧光抗体技术.用火焰、甲醇、丙酮三种方法固定标本,分别经过10 min和20 min两种不同时间固定,然后滴加不同稀释倍数的兔抗血清,滴加羊抗兔IgG-FITC,于荧光显微镜下观察.结果表明甲醇固定10 min和火焰固定,兔抗血清抗体效价为1:80时效果最好.     

鸡传染性喉气管炎病毒PCR诊断方法的建立与应用

根据GenBank登录的传染性喉气管炎病毒(ILTV)的TK基因序列设计并合成1对特异性引物,以ILTV疫苗株DNA为模板,建立了检测ILTV TK基因的PCR方法。应用该方法能从临床分离毒株和疫苗株中扩增到长为427 bp的目的片段;但不能从新城疫病毒(NDV)、传染性法氏囊病毒(IBDV)、禽呼肠孤病毒(ARV)、减蛋综合征病毒(EDSV)、H9亚型禽流感病毒(H9-AIV)、传染性支气管炎病毒(IBV)、大肠杆菌以及金黄色葡萄球菌等病原中扩增出阳性条带;敏感性试验表明其DNA最小检出量为4.9 ng;应用该方法和病毒分离法对2份临床病例和人工感染鸡的检测,两者符合率为100%。上述结果表明该PCR方法具有良好的特异性和敏感性,可用于传染性喉气管炎病毒鉴定和临床诊断。