Comparison of enzyme-linked immunosorbent assays and virus neutralization test for detection of antibodies to avian pneumovirus

Two different whole-virus enzyme-linked immunosorbent assays (ELISAs), developed in Ohio (OH) with APV/Minnesota/turkey/2a/97 and in Minnesota (MN) with APV/Colorado/turkey/97, and the virus neutralization (VN) test were used to test 270 turkey serum samples from 27 Minnesota turkey flocks for avian pneumovirus (APV) antibodies. In addition, 77 turkey serum samples and 128 ostrich serum samples from Ohio were tested. None of the turkey samples from Ohio had antibodies to APV by the VN test and OH ELISA. The ostrich samples were only tested with the VN test and were all negative for antibodies to APV. For the Minnesota serum samples, 107, 115, and 120 were positive by the VN test, the OH ELISA, and the MN ELISA, respectively. The Kappa values of 0.938 and 0.825 showed excellent agreement between the VN test and the OH ELISA and the MN ELISA, respectively, for detection of antibodies to the APV. The OH ELISA and MN ELISA had sensitivities of 1.0 and 0.953, specificities of 0.950 and 0.889, and accuracies of 0.970 and 0.914, respectively. Our results indicate that the 3 methods are sensitive and specific for diagnosis of the APV infection.     

Serologic comparison of avian reovirus isolates using virus neutralization and an enzyme-linked immunosorbent assay

The serologic relatedness of six avian reovirus isolates (CO8, S1133, 81-5, 2408, 1733, and UMI 203) were determined using a virus-neutralization (VN) test and an enzyme-linked immunosorbent assay (ELISA). Six groups of 20 specific-pathogen-free broilers each were twice infected with one of the six isolates per group. Serum was reacted against each isolate in a beta VN test in chicken embryo kidney cells and against the S1133 virus in ELISA. Relatedness (R) values, determined by cross VN, revealed that all belonged to a single serotype. However, the CO8 isolate represented a major subtype difference compared with the other isolates. R values among the five other isolates indicated minor or no subtype differences. ELISA also showed major differences between the CO8 and the other isolates.     

Serological and virological studies of Newcastle disease and avian influenza in slaughter-age ostriches (Struthio camelus) in Japan

Serum samples from 191 ostriches (Struthio camelus) in Japan were tested for antibodies to Newcastle disease virus (NDV) and avian influenza virus (AIV). Twenty-two (12%) contained NDV-specific neutralizing antibodies by a virus-neutralization (VN) test without vaccination. Antibodies to AIV were not detected in the any sera by an agar gel precipitation test. Seven serum samples that had vaccinated with live NDV by eye drop were all positive by the VN test at 1 month post vaccination. A haemagglutination inhibition (HI) test for NDV seemed not to be suitable for ostriches because of non-specific agglutination of chicken red blood cells. No haemagglutinating viruses were isolated. This is the first report on detection of antibodies against NDV in ostriches in Japan.     

H9N2亚型禽流感病毒抗原性变异的研究

对1998—2002年间在河南省豫北地区分离到的5株H9N2亚型禽流感病毒的抗原性变异进行了研究。经HI试验、鸡胚中和试验、细胞中和试验及攻毒保护试验证明，5株H9N2亚型间已经发生了抗原性漂移。98A5和99S毒株间的保护力接近100％，HI试验、鸡胚中和试验、细胞中和试验的相关性均在0．74以上。表明2毒株间的抗原性相近；用00Y毒株攻击其他4株免疫的鸡，其保护率仅为60％～80％；而02Y株对除00Y株外的4株的免疫保护率分别为60％、75％、80％、100％，与分离年代呈负相关性，HI、鸡胚中和试验、细胞中和试验也取得类似结果，说明2000年后的毒株间已发生抗原性变异。     

Development and preliminary application of an indirect ELISA for detecting antibodies against Avian Influenza Virus

Fragment of 759 bp DNA spanning the Matrix 1 (M1) gene of Avian Influenza Virus (AIV) was inserted into an expression vector pET28c to construct a recombinant plasmid pET28c-M1. The pET28c-M1 plasmid was transformed into the Escherichia coli BL21 (DE3) competent cell to produce a recombinant strain E. coli 21 (DE3). After being induced by Isopropyl-b-D-galactopyranoside (IPTG), E. coli 21 (DE3) expressed a 28-kDa fusion protein at a high level. This protein can bind anti-AIV (H5N1) positive serum by Western-blot analysis. After being denatured, renatured, and purified by Ni(2+)-column, the fusion protein was used as an antigen to develop Matrix 1 Enzyme-Linked Immunosorbent Assay (M1-ELISA) for detecting antibodies against AIV from chicken serum. We found that this indirect M1-ELISA was sensitive for differentiating antisera against AIV and antisera against other six kinds of avian viruses apart from AIV and this method is more sensitive than Hemagglutination Inhibition (HI) test. When compared with HI test and ELISA (IDEXX) in evaluating 581 serum samples from field vaccinated chickens, this assay showed 93.3% agreement ratio with the HI test, as well as 96.0% agreement ratio with ELISA (IDEXX). In a preliminary application, the assay successfully detected 19 AIVs from 51 nonvaccinated chicken lungs. It concludes that an indirect ELISA was successfully developed for detecting AIV. The assay is specific and sensitive. The application will greatly contribute to the long-term prevention and control of avian influenza in China.     

Detection of antibodies against serotypes 1 and 2 infectious bursal disease virus by commercial ELISA kits

Two distinct serotypes of infectious bursal disease virus (IBDV) are recognized in chicken and turkey flocks in the United States. Serologic testing of chicken flocks for serotype 1 viruses is routinely performed to monitor disease status and vaccination. Earlier studies indicated that enzyme-linked immunosorbent assay (ELISA) test detects antibodies to both serotypes of the virus, while the virus neutralization (VN) test is serotype specific. It is useful to evaluate currently available commercial ELISA kits for their ability to differentiate between antibodies elicited by the two serotypes. Three trials were performed in which chickens were orally inoculated with either a high or a low dose of serotype 1 STC or serotype 2 OH strains of IBDV. Sera collected at 0, 7, 14, and 21 days from these chickens and antisera procured from naturally infected broiler (n=20) and layer (n=30) flocks were tested with five different commercial ELISA kits and by VN. All ELISA kits detected different levels of antibodies elicited against serotype 1 of the virus and moderate and high levels of antibodies against serotype 2 virus. A correlation existed between the ELISA and the VN titers of experimentally infected chickens. All serum samples tested from the commercial layer flocks and 65% of the broiler flocks had antibodies against the OH strain. However, no correlation between the VN titers and ELISA titers was observed for the commercial broilers and layers sera by the majority of the kits. The results indicated that currently available commercial ELISA kits detect antibodies elicited by the two serotypes of IBDV. Hence, the prevalence of serotype 2 antibodies in the flocks should be considered while determining antibody profiles of the flocks against serotype 1 viruses.     

Antigenic cross-reactivity among avian pneumoviruses of subgroups A,B, and C at the matrix but not nucleocapsid proteins

Earlier findings from our laboratory based on analysis of nucleotide and predicted amino acid sequence identities of 15 avian pneumoviruses (APVs) isolated from the United States (subgroup C) demonstrated that the viruses were phylogenetically separated from the European subgroup A and subgroup B viruses. Here, we investigated whether viruses from the three subgroups were cross-reactive by testing field sera positive for each of the APV subgroups in an enzyme-linked immunosorbent assay (ELISA) test with recombinant matrix (M) and nucleoprotein (N) proteins generated from a Minnesota APV isolate (APV/MN2A). Sera from turkeys infected with APV subgroup A, B, or C reacted with recombinant M protein derived from APV/MN2A. In contrast, recombinant N protein from APV/MN2A virus was reactive with sera from subtypes A and C viruses but not from subtype B virus. The results illustrate that viruses from the three APV subtypes share antigenic homology, and the M protein-based ELISA is adequate for monitoring APV outbreaks but not for distinguishing between different subtypes.     

抗H5亚型禽流感病毒血凝素蛋白特异性单克隆抗体的研制及鉴定

以抗H5亚型禽流感病毒（AIV）JSD株鸡胚尿囊液免疫8周龄BALB/c小鼠,第4次免疫后取其脾淋巴细胞与SP2/0-Ag-14骨髓瘤细胞融合,用血凝抑制试验（HI）对杂交瘤进行筛选,经3次亚克隆后,共获得4株针对血凝素（HA）蛋白的特异性单克隆抗体,分别命名为：2D2、2C8、3D3和3D11。该4株单抗的小鼠腹水的HI效价在2^13～2^15,ELISA效价为1.2×10^5～5.1×10^5。亚类鉴定结果表明,2D2和3D11属于IgG1,2C8属于IgG3,3D3属于IgG2a亚类;特异性试验结果表明,上述4株单抗仅与H5 AIV毒株发生特异性反应,而不与其他亚型AIV以及NDV、IBV和EDSV-76等病毒反应;鸡胚中和试验结果显示,4株单抗均具有较好的中和活性。本研究成功获得4株针对H5 AIVHA蛋白的特异性单抗,为临床H5 AIV的血清学监测及鉴定提供了必需的试剂。     

Comparative serological evaluation of avian influenza vaccine in turkeys

Four- and six-week-old turkeys were vaccinated subcutaneously using avian influenza virus (AIV) A/Duck/613/MN/79 (H4N2) killed oil-emulsion vaccine. Sequential serological tests using agar gel precipitin (AGP), hemagglutination inhibition (HI), and enzyme-linked immunosorbent assay (ELISA) for measuring antibodies to AIV were performed up to 4 weeks postvaccination, when birds were challenged intranasally using A/Turkey/MN/80 (H4N2) live AIV. The ELISA was 25 to 1600 times more sensitive than the HI test and was able to detect antibody production earlier than the HI test. All turkeys with an ELISA titer of greater than or equal to 800 were protected against homologous challenge, as measured by virus recovery 3 days postchallenge. Four turkeys out of 20 serologically negative by AGP and HI tests but ELISA-positive were protected.     

Serologic detection of the occurrence of avian paramyxoviruses in pigeons

447 blood-serum samples of racing and free living pigeons collected in 11 districts of Czechoslovakia from August 1983 till March 1984 were examined by the haemagglutination inhibition test to the Newcastle disease virus, strain Roakin, to the pigeon PMV-1 and to the PMV-3; 121 of the samples were tested to other serotypes, PMV-2--PMV-9, and to the avian influenza A virus. 58.4% of samples were positive (greater than or equal to 2 log2) to the Roakin strain with the mean titre 3.6 log2 and 65.1% to the pigeon PMV-1 with the mean titre 4.5 log2. All samples tested were negative to other serotypes except two samples of one group positive to PMV-8 with the mean titre 4.3 log2. The titres of HI antibodies to the Roakin strain and to the pigeon PMV-1 were compared. The risk of the transmission and of the readaptation of pigeon virus to poultry was discussed.     

间接ELISA检测禽流感抗体方法的建立

应用醛化的鸡红细胞经吸附释放方法获得纯化的禽流感病毒 (AIV) ,经 triton X-1 0 0处理并反复冻融制备禽流感 EL ISA抗原。应用抗鸡 Ig轻链单抗 1 B7辣根过氧化物酶结合物建立了定量检测禽流感抗体水平的 EL ISA方法。确立了将鸡血清 1 0 0倍稀释监测 EL ISA效价(ET)的回归方程 y=0 .77452 x 0 .8793 5(r=0 .9466) ,可用于定量测定。血凝抑制 (HI)抗体与 EL ISA方法的比较试验表明 ,EL ISA法比血凝抑制试验敏感 1 .5倍以上     

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.     

An enzyme-linked immunosorbent assay for detection of antibodies to exogenous avian leukosis virus

A microplate enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to avian leukosis virus (ALV) of subgroups A and B in infected chickens was developed with the use of Rous-associated virus (RAV)-1 (subgroup A) and RAV-2 (subgroup B) antigens purified by sucrose-gradient centrifugation. The antigen was used for ELISA after treatment with Triton X-100. In the ELISA, the subgroup viral antigen reacted strongly with homologous antiserum but also reacted with heterologous antiserum. Tests with serum absorbed with purified homologous and heterologous virus and tests for antigen-blocking by group-specific antibodies to ALV revealed that the reaction was caused mainly by subgroup-specific antibodies. The ELISA was 8 to 32 times more sensitive than the virus-neutralization (VN) test and detected antibodies to ALV earlier than the VN test in chickens infected experimentally with RAV-1 and RAV-2. In field application of the ELISA, 44.2% of 484 chicken sera were positive for RAV-1 and/or RAV-2 antigen, and 80.4% of flocks were positive. These findings indicate that ELISA is superior to the VN test in sensitivity, simplicity, rapidity, and applicability for large-scale field surveys for ALV infection.     

Development of enzyme-linked immunosorbent assay with nucleoprotein as antigen for detection of antibodies to avian influenza virus

During the avian influenza outbreak of 2003-04 in Southeast Asia, two avian influenza viruses (AIV), one of H5N1 subtype and the other H9N2 subtype, were isolated and identified from local farms. The nudeoprotein (NP) gene of the H5N1 AI isolate was cloned, and the segment encoding amino acid 47-384, which covers its major antigenic domains, was subcloned and expressed in E. coli. Subsequently, the NP (47-384) expression product was purified and used as the diagnostic antigen to develop a NP-based type-specific indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to AI from chicken sera. The ELISA is shown to be specific for AIV and does not cross-react with chicken sera that has antibodies to other avian viruses. The NP(47-384)-ELISA was compared with a hemagglutination inhibition test and a commercial AIV ELISA kit in evaluating 150 sera samples from experimentally AIV-infected or vaccinated specific-pathogen-free (SPF) chickens. Our NP(47-384)-ELISA was more sensitive than the two tests and showed an 82% agreement ratio with the HI test and an 80.67% agreement ratio with the commercial kit. The NP(47-384)-ELISA and the commercial AIV ELISA were used to evaluate 448 field sera samples from diseased chickens or vaccinated chickens during the 2003-04 AI outbreak in China. The two ELISA tests had a 95% agreement ratio. We conclude that the NP(47-384)-ELISA developed in our laboratory was specific and sensitive and it has great application potential in China's long-term prevention and control of AI.     

Seroprevalence of avian influenza virus, infectious bronchitis virus, reovirus, avian pneumovirus, infectious laryngotracheitis virus, and avian leukosis virus in Nigerian poultry

Eight poultry farms in Nigeria, including chickens from nine breeder, 14 broiler, 28 pullet, 11 layer, and three cockerel flocks, were tested for antibody seroprevalence to the following poultry viruses of potential economic importance: infectious bronchitis virus (IBV), avian reovirus, avian pneumovirus (APV), infectious laryngotracheitis virus (ILTV), avian influenza virus (AIV), and avian leukosis virus (ALV). Serum samples were collected between 1999 and 2004 and were tested for antibodies using commercial enzyme-linked immunosorbent assay (ELISA) kits. Seroprevalence was very high for IBV (84%); intermediate for reovirus (41%), APV (40%), and ILTV (20%); and very low for ALV (<5%) antibodies. By commercial ELISA, the seroprevalence of antibodies against AIV was, in some flocks, up to 63%. However, more specific assays did not confirm AIV antibodies, indicating that all flocks tested were free of avian influenza antibodies. Birds seemed to be first infected by IBV (at about 7 wk of age), then by reovirus at 12 wk, before they became infected by APV (week 25) and ILTV (week 30). This is the first report of serological evidence of the above viruses in West Africa. Further studies are necessary to assess economic losses due to these avian viruses and the costs and benefits of countermeasures.     

Detection of antibodies to avian pneumovirus by a micro-indirect immunofluorescent antibody test.

A micro-indirect immunofluorescent antibody (micro-IFA) test with a 96-well, flat-bottomed microplate was developed for measuring avian pneumovirus (APV) antibodies. Two Japanese APV strains (MM-1, 8597/CV94) isolated at different places and times and Vero cells were used for antigen preparation in this test. The test results were compared with those of a serum neutralization (SN) test. By the micro-IFA test, specific immunofluorescent antigens were observed in the cytoplasm of cells infected with either strain, and the antibody titers of antisera to these strains were quite similar. In most cases, the results were obtained within 3 hr. Antibody titers between the micro-IFA and SN tests were highly correlated, with correlation coefficients of 0.873 (MM-1 strain) and 0.889 (8597/CV94 strain). We also investigated APV antibody status in two farms for a period of about 2 yr by the micro-IFA test and revealed that APV infections were repeated within these farms. On the basis of these results, we conclude that our micro-IFA test is useful for routine serologic surveys of APV infections, particularly when a large number of samples are to be treated, because this test was time and labor saving relative to SN tests or conventional IFA tests utilizing embryo tracheal organs or coverslip cell cultures.     

Serologic and pathogenetic studies on avian reoviruses isolated in Japan

Eighty-nine avian reoviruses isolated from diseased and clinically normal chickens were classified serologically using antisera against five prototype strains. Eighty-three strains were classified into five serotypes; six strains were untypable. Most of the cytopathogenic strains that produced a clear cytopathic effect (CPE) in chicken embryo fibroblasts (CEFs) were highly pathogenic for chicken embryos (80% or more mortality via the allantoic sac) and for chicks (severe footpad swellings and tenosynovitis). These strains were classified into a single serotype represented by the TS-142 prototype strain. However, 10 strains that could not produce a clear CPE in CEFs showed very low pathogenicity for embryos and chicks, and these strains were serologically different from the TS-142 prototype strain. There was a strong relationship between pathogenicity and serotype. About 17% of the isolates were considered highly pathogenic.     

Antigenic comparisons of selected avian reoviruses by use of the plaque-reduction neutralization assay

The antigenic interrelatedness of 3 clone-purified turkey reoviruses (NG-Turkey, 82-88, and NC-TEV) to each other and to 4 clone-purified chicken reoviruses (S1133, Co8, Fahey-Crawley, and avian type 2) was determined in reciprocal cross-neutralization tests, using polyclonal antisera and the plaque-reduction technique. The morphologic features of plaques formed under agar were studied for all 7 reoviruses, and size comparisons for turkey vs chicken isolates were made. All 3 turkey reoviruses (with the exception of NG-Turkey vs Fahey-Crawley chicken reovirus) formed plaques significantly (P less than 0.05) smaller than plaques produced by their chicken counterparts. The 3 turkey reoviruses were closely related to each other and to chicken reovirus CO8. The antigenic differences between turkey reoviruses 82-88 and NC-TEV and chicken reovirus S1133 were slight (minor subtype); however, the latter and NG-Turkey were serotypically distinct. The NG-Turkey and 82-88 turkey reoviruses were more related (minor subtype) to the Fahey-Crawley and avian type 2 chicken reoviruses, than was NC-TEV turkey reovirus (major subtype).     

三种检测禽副粘病毒2型(PMV-2)抗体方法的建立及其与HI方法的比较

建立了检测血清中禽副粘病毒2型（PMV-2）抗体的间接免疫荧光试验（IFA）、间接法酶联免疫吸附试验（ELISA）以及斑点酶联免疫吸附试验（Dot—ELISA）方法，并将三种方法同血凝抑制试验（HI）进行了比较。结果表明，建立的IFA、间接ELISA和Dot—ELISA等三种抗体检测方法其敏感性、特异性均很好，与HI方法相比，敏感性也大大增强。     

Efficacy of oil-emulsion vaccines prepared with pigeon paramyxovirus-1, Ulster, and La Sota Newcastle disease viruses

Three strains of avian paramyxovirus-1 virus (PMV-1) were used to prepare four experimental monovalent oil-emulsion vaccines. A pigeon PMV-1 isolate (PPMV-1) and the Newcastle disease virus strains La Sota and Ulster were used to prepare four pools of beta-propiolactone-inactivated allantoic fluid for the vaccines. Groups of susceptible white rock chickens and racing homing pigeons were vaccinated subcutaneously with one of the vaccines, and their serologic responses were determined using the hemagglutination-inhibition (HI) test at frequent intervals up to 9 weeks postvaccination. Pigeons were challenged after 10 weeks with a virulent PPMV-1 isolate given intravenously, observed for signs of disease for 5 weeks, and then tested for secondary serologic HI responses. The HI responses were measured using the three strains of virus as HI test antigens. The titers were generally greater when the hemagglutination antigen used in the test was homologous with the antigen used to prepare the vaccine. All vaccines protected pigeons against morbidity and death but not against infection with the challenge virus. The shedding of PPMV-1 challenge virus from PPMV-1 vaccinates was greatly reduced 6 days after challenge.