Antibody responses against avian reovirus nonstructural protein sigmaNS in experimentally virus-infected chickens monitored by a monoclonal antibody capture enzyme-linked immunosorbent assay

Crude antigen preparations from avian reovirus (ARV)-infected chicken embryo fibroblasts (sigmaNS) or from bacterially expressed protein sigmaNS (esigmaNS) were captured by monoclonal antibody 1E1(MAb 1E1) against ARV nonstructural protein sigmaNS immobilized on the ELISA plates and were used as the MAb capture ELISA for antibody detection. Sixty one-week-old specific pathogenic free (SPF) chickens were divided into six groups and were vaccinated with live or inactivated ARV vaccine preparations in different combinations or inoculated with a virulent ARV strain. Sera collected from the birds were tested for their antibody responses to ARV nonstructural protein sigmaNS. Using the MAb capture ELISAs, the level of nonspecific binding reactions was tested on the serum samples obtained weekly from mock-infected SPF chickens from 1 to 25 weeks and compared to the results tested by the conventional ELISA. The results indicated that both MAb capture ELISAs had lower nonspecific bindings than those in the conventional ELISA, even in older birds. Antibody responses against ARV sigmaNS of the birds which received the inactivated vaccine twice (group I), inactivated vaccine followed by a live vaccine (group II), or a live vaccine followed by boosting with an inactivated vaccine (group III) were detected by MAb captured ELISA with sigmaNS crude antigens. The absorbance values increased rapidly at 1-2 weeks after boosting, approximated a peak at 5-6 weeks of age, and maintained this throughout the length of the experiment. The absorbance values of the MAb capture ELISA showed a good correlation to the SN titers ( r value > 0.85). On the other hand, serum samples from the birds which received the live vaccine twice (group IV) or were inoculated with a virulent ARV (group V) did not show antibody responses to sigmaNS, similar to those from the mock-infected birds (group VI), as the absorbance values maintained at a low level (below 0.5) throughout the length of the experiment. Similar results were obtained in the sera detected by MAb capture ELISA with crude esigmaNS antigens, except that the absorbance values in the sera from the birds in group III were gradually increased and later approximated a peak at 11 weeks of age and maintained this throughout the length of the experiments. The results suggest that MAb capture ELISAs can be readily used to detect antibody responses of the birds against ARV nonstructural protein sigmaNS which may reflect an immune status of a chicken flock, receiving ARV vaccine as long as including an inactivated vaccine.     

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.     

An enzyme-linked immunosorbent assay with the recombinant merozoite protein as antigen for detection of antibodies to Eimeria necatrix

A cDNA library was constructed with Eimeria necatrix merozoite mRNA and immunologically screened by chicken sera against this parasite. One of the positive clones containing an insert of 879 nucleotides, pNP19, showed similarity to part of a published gene expressed in E. tenella merozoite by the homology search system. The inserted DNA was subcloned into baculovirus, and a 35-kD protein was expressed, purified, and used for the antigen in enzyme-linked immunosorbent assay (ELISA). Antibodies from the chickens vaccinated with the E. necatrix attenuated strain, Nn-P125, were detected from 14 days after vaccination by ELISA. The mean absorbance increased rapidly to a peak around 21 days after vaccination; thereafter, it began to decline. Even though some of the vaccinated chickens showed very low levels of antibody response to the recombinant protein 56 days after vaccination, they were protected against challenge with virulent strain of E. necatrix. The mean absorbances in sera from both vaccinated and nonvaccinated chickens highly increased 14 days after challenge. On the other hand, the antibody was not detected in ELISA when chickens were exposed to other Eimeria species such as E. tenella, E. acervulina, and E. maxima. These results demonstrate that this recombinant protein is suitable for detecting the specific antibody in chickens infected with both attenuated and virulent strains of E. necatrix.     

A standardized enzyme-linked immunosorbent assay for infectious bronchitis virus: comparison with hemagglutination-inhibition and virus-neutralization assays for measuring protective antibody levels in chickens

An enzyme-linked immunosorbent assay (ELISA) was developed to measure antibodies to infectious bronchitis virus (IBV) in chickens. The results are reported in IBV standard ELISA values calculated by comparing antibody levels in test sera with antibody levels in a series of standard reference sera. The IBV standard ELISA values were good indicators of responses to vaccination and the immune status of experimentally challenged birds. Although the assay was not serotype-specific, the sensitivity makes it ideally suited for determining the immune status of poultry flocks. The assay results compared favorably with other laboratory results, including virus-neutralization titers, hemagglutination-inhibition levels in sera, virus isolation from vaccinated/challenged birds, and the tracheal ring test results.     

An enzyme-linked immunosorbent assay for coccidiosis in chickens: correlation of antibody levels with prior exposure to coccidia in the laboratory and in the field

An enzyme-linked immunosorbent assay was developed for detecting antibody to coccidia to facilitate the survey of laboratory and field infections. Serum antibody levels in chickens were measured against soluble Eimeria tenella oocyst antigen. Sera from breeders aged 10, 23, 37, and 43 weeks were positive with uniformly high antibody titers. Broiler chick sera showed high maternal antibody titer at hatch, decreasing to an almost negligible response at 3 weeks of age. Two-week-old broiler chicks had variable responses to a single infection of E. tenella: titers were elevated at 8 to 10 days postinfection and generally increased through day 24. Weekly reinfection of 2-week-old broiler chickens produced an antibody titer in proportion to the number of oocysts per dose and stimulated protection against challenge with 2 x 10(5) E. tenella. Inbred birds raised in a pathogen-free environment for 6 weeks had no detectable antibody titers.     

应用单克隆抗体酶联免疫斑点试验检测抗传染性喉气管炎抗体

本文提出以硝酸纤维膜作为固相载体，辣根过氧化物酶标记的抗传染性喉气管炎病毒（ＩＬＴＶ）的单克隆抗体（ＭｃＡｂ），邻苯二胺为底物，检测ＩＬＴＶ抗体的免疫斑点试验方法（Ｄｏｔ－ＥＬＩＳＡ）。该方法较常规方法敏感，特异、省时。该方法的建立为鸡群疫病监测，免疫鸡群抗体水平的检测提供了新的可靠方法。     

A monoclonal antibody-blocking enzyme-linked immunosorbent assay for the detection of serovar-specific antibodies to Haemophilus paragallinarum

Two monoclonal antibody-blocking enzyme-linked immunosorbent assays (B-ELISAs) were developed to detect serovar-specific antibodies to Haemophilus paragallinarum. One assay detected antibodies against serovar A and the other antibodies against serovar C. The assays were evaluated with sera derived from disease-free chickens as well as chickens experimentally immunized and/or challenged with H. paragallinarum strains 0083 (serovar A), Modesto (serovar C), or HP31 (serovar C). When tested with 440 negative sera (170 from a specific-pathogen-free and 30 from each of nine commercial layer flocks), both tests gave only a single false-positive reaction. The use of the B-ELISAs with the experimentally produced sera showed the assays to be serovar specific. With the exception of one serum, the serovar A B-ELISA detected antibodies only in the chickens vaccinated with 0083. Similarly, with the exception of one serum, the serovar C B-ELISA detected antibodies only in those chickens vaccinated with Modesto or those chickens challenged with HP31. Overall, the serovar A B-ELISA had a specificity of 99.7% and a sensitivity of 78.7%, whereas the serovar C B-ELISA had a specificity of 99.8% and a sensitivity of 64.7%.     

A blocking ELISA for the detection of specific antibodies to bovine respiratory syncytial virus

A blocking enzyme-linked immunosorbent assay (ELISA) has been adapted to detect specific antibodies in bovine sera to respiratory syncytial virus using a horseradish peroxidase-labeled monoclonal antibody to the fusion protein of the virus. This assay plus an indirect blocking ELISA and indirect ELISA were used to detect antibodies to the bovine respiratory syncytial virus (BRSV) in 159 field-origin bovine sera. Results of these assays were compared with serum antibody titers measured by the serum neutralization (SN) test. Over a 56-day period, the mean neutralization titers and the mean delta absorbance values for the blocking ELISA, on the same sera, showed similar declines. However, the calculated correlation coefficients between mean SN titer and mean absorbance value for the blocking ELISA of the individual sera ranged from -0.2 to -0.5 depending on the source of sera. Similar values were obtained whether using crude or purified viral antigen in the assays. Corresponding calculated correlation coefficients were generally higher for the indirect blocking ELISA or indirect ELISA than for the blocking ELISA. The blocking ELISA was between 70 and 64% as sensitive as the serum neutralization test with a specificity of 100 or 90% using the crude and purified viral antigen, respectively. The indirect blocking ELISA and indirect ELISA had similar calculated sensitivities and specificities. The blocking ELISA was faster to run than either of the other ELISA's or the neutralization test. Further, nonspecific background absorbance was obviated because the blocking ELISA detects antibodies to 1 specific viral protein, the fusion protein. These studies suggest that the blocking ELISA should be useful as a serological test for BRSV antibodies.     

Cell-culture virus-neutralization test and enzyme-linked immunosorbent assay for evaluation of immunity in chickens against fowlpox

Two serological tests--the virus-neutralization (VN) test in chicken embryo fibroblasts (CEF) using a cell-culture-adapted virus, and the enzyme-linked immunosorbent assay (ELISA)--were used for evaluating the immune response in chickens against fowlpox virus. The VN test was conducted in 96-well tissue-culture plates using a fowlpox virus that was adapted to induce cytopathic effects (CPE) in CEF in 48 hr. The ELISA was carried out with an antigen prepared by precipitation of a cell-culture-propagated virus suspension with ammonium sulfate and concentration by centrifugation. A 0.1 M acetate buffer, pH 5, was used as the sensitizing solution for maximum specific binding of the antigen to the microplate plastic well. No antibodies were detected by the VN test in 228 serum samples taken from chickens at irregular intervals between 1 and 39 weeks of age, even though the birds were vaccinated against fowlpox at 13 weeks of age. However, in sera collected 4 weeks after a sample of laying hens was challenged with fowlpox virus, VN titers of 1/10 to 1/40 were detectable. On the other hand, significant antibody reactions were detected by the ELISA on sera from chickens during the growing period, following vaccination and challenge. Although no maternal antibodies were found at 1 week of age, a continuous increase in the mean ELISA titers to fowlpox was demonstrated during the entire experimental period. This study showed that the ELISA was considerably more sensitive and practical than the VN test.     

Development of a blocking enzyme-linked immunosorbent assay for detection of turkey antibodies to Mycoplasma meleagridis

A blocking enzyme-linked immunosorbent assay (B-ELISA) was developed to detect antibodies to Mycoplasma meleagridis (MM) in turkey sera. This assay was based on two mouse monoclonal antibodies recognising all MM strains tested but none of seven avian mycoplasmal species tested. Furthermore, their binding to the Tween 20 antigen was inhibited by serum from MM-infected birds. The B-ELISA test format was optimized. The cut-off was determined using a set of sera from MM-free turkeys. This B-ELISA was then compared with a commercial indirect ELISA (I-ELISA). Specificities of the two ELISA tests were not significantly different (100 or 99%, respectively). The sensitivity of B-ELISA was significantly higher than the I-ELISA when I-ELISA suspicious results were considered as negative. Testing sera from experimentally MM-infected animals showed that serum plate agglutination (SPA) test detected positive birds before both ELISA methods. Samples were collected in MM-infected commercial flocks and analyzed by SPA, ELISAs, MM-PCR or culture. Results showed that the sensitivity of the B-ELISA appeared superior to the I-ELISA. Moreover, the ability to detect maternal antibodies makes it a useful tool for eradication or control of MM infections.     

Investigations on the Influence of Helminth Parasites on Vaccination of Chickens against Newcastle Disease Virus under Village Conditions

Prevalence studies have shown that almost 100% of free-range chickens are infected with a wide range of parasites. The infections are mostly subclinical in nature, resulting in production losses and occasionally mortality. Newcastle disease (ND), on the other hand, results in high mortality rates during epidemics. ND is a limiting factor for increasing poultry production in many tropical countries, where frequent reports indicate vaccination failures. The aim of our study was to investigate the influence of helminths on the antibody response after vaccination against Newcastle disease of free-range chickens naturally infected with parasites. Sixty chickens were divided into six groups, of which three were vaccinated against ND with a live De Soto vaccine, while the other three remained non-vaccinated. One group within the vaccinated groups and the one within the non-vaccinated group was kept naturally infected with helminth parasites, while the other two groups in each set were dewormed with fenbendazole and niclosamide, and one of each of these groups was subsequently infected with Ascaridia galli. After vaccination, all the groups were followed for 5 weeks and their antibody titres were determined weekly using a HI test. All the birds were finally challenged 4 weeks after vaccination with a virulent velogenic ND virus obtained from a field outbreak. All the vaccinated chickens seroconverted and had high antibody levels after 3 weeks, but these dropped to low levels at 4 weeks after vaccination. After challenge, the antibody titres rose in the dewormed groups but not in the parasite-infected groups. After 5 weeks, all the parasite-infected animals had significantly lower antibody titres than the dewormed animals. All the vaccinated chickens survived the challenge infection, emphasizing the importance of the cellular immune response. Further studies are needed to examine the effects of the parasitic infection on protection against ND over a longer period.     

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.     

Blocking enzyme-linked immunosorbent assay for detection of antibodies against bovine enterovirus

A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against bovine enterovirus (BEV) in bovine sera. In this ELISA, bovine serum samples were allowed to react with captured viral antigens (by specific chicken IgG), before the addition of specific mouse IgG for measuring non-occupied viral epitopes. The ELISA was slightly more sensitive and required a shorter time period than traditional serum neutralisation (SN). Among the 871 bovine serum samples tested so far, the titres produced by this assay had a significant correlation with those recorded by SN. The ELISA could be used as an alternative assay for SN in a large-scale BEV antibody investigation.     

检测鸡球虫四价弱毒疫苗抗体应答的ELISA方法的建立及初步应用

本研究旨在建立检测鸡球虫四价弱毒疫苗(柔嫩艾美耳球虫、毒害艾美耳球虫、堆型艾美耳球虫、巨型艾美耳球虫)抗体应答的酶联免疫吸附试验(ELISA),并初步阐明鸡免疫四价弱毒疫苗及攻虫后的抗体应答。将300羽岭南黄鸡分为A(免疫攻虫组)、B(不免疫不攻虫组)、C(不免疫攻虫组)共3个组。A组雏鸡在4日龄时进行首免,11日龄时鸡开始二免,二免完成后(17日龄时)对A组及C组的鸡进行攻虫(28万个4种球虫的混合卵囊/羽),7 d后检查A、C两组鸡的存活率。在鸡二免和三免完成后,分别对A、B组进行采血,通过优化最佳抗原包被浓度、酶结合物工作浓度及最佳血清稀释度,建立了ELISA方法来检测鸡体对球虫四价弱毒疫苗的抗体应答情况。A组鸡血清抗体的平均D值为0.870和0.904,明显高于B组鸡的平均D值0.261和0.270,证明了鸡体免疫疫苗后可以刺激产生相应抗体。免疫鸡攻虫后的存活率和抗体应答的检测结果,都充分说明了此鸡球虫弱毒四价疫苗具有良好的免疫原性,所建立的ELISA方法为今后评价鸡球虫弱毒四价疫苗的免疫效力提供了有效手段。     

Relationship of the enzyme-linked immunosorbent assay to indirect immunofluorescent antibody test for the detection of so-called chicken anemia agent antibodies in serum from broiler breeders.

Serum samples collected from breeder chickens ranging in age from 1 day to 55 weeks were tested for CAA antibodies by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent antibody (IFA) test. The relationship of ELISA to IFA test was determined. The sensitivity of the ELISA relative to the IFA test was 82.64%, and the specificity of the ELISA relative to the IFA test was 56.25%. Agreement between the ELISA and the IFA test was highly significant (Kappa = 0.74, Z = 5.78). We concluded that the ELISA is as good as the IFA test for detecting CAA antibody in sera from chickens.     

Seroprevalence of Histomonas meleagridis in pullets and laying hens determined by ELISA

Serum samples from 1120 layers from 56 flocks and 400 pullets from 20 flocks were tested by an indirect sandwich ELISA to investigate the prevalence of antibodies to Histomonas meleagridis in chickens kept in alternative husbandry systems. The overall prevalence of antibodies to H meleagridis in layers was 37.3 per cent, and positive birds were identified in 50 flocks. This was significantly higher than in pullets, where only 8.3 per cent of the birds tested positive. Optical density (OD) values obtained from pullet sera were much lower than the OD values from layers; however, positive birds were detected in half of the pullet flocks. In particular, all birds from an organic pullet flock were found to be positive, with high OD values. Overall, the highest prevalence of positive sera was obtained from birds kept in free-range flocks. Attempts to reisolate live histomonads from birds in 18 layer flocks were unsuccessful.     

副鸡嗜血杆菌人工感染鸡抗体动态的研究

本研究采用阻断ELISA（B－ELISA）和血凝抑制试验（HI）方法,检测了鸡体分别人工感染副鸡嗜血杆菌Hp8株（A型）和H668株（C型）后0－9周的血清抗体消长情况,并绘制了示意抗体曲线。结果表明,Hp8人工感染后,从第2周到第9周,B－ELISA阳性检出率一直保持100％,其中抗体效价在感染后第4周达到效价高峰,平均约为15．2。而H688人工感染鸡则在感染后第7周检出率最高,此后急剧下降。AI方法对A型抗体的阳性检出率亦在在第4－5周达到高峰,而对C型抗体的检出率一直较低。结果进一步显示了B－ELISA良好的特异性和敏感性,为本方法及其试剂盒产品的进一步应用提供了参考。     

An enzyme-linked immunosorbent assay for the detection of bovine antibodies to vesicular stomatitis virus

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect bovine antibody to vesicular stomatitis virus (VSV). Serum samples from cows experimentally infected with the New Jersey serotype of VSV (VSV-NJ) were assayed by the ELISA and serum-neutralization (SN) assay. The ELISA was as sensitive as the SN assay in detecting bovine antibody to VSV. The correlation between SN titers and ELISA values at absorbance at 405 nm was statistically significant. The ELISA was not specific for VSV-NJ, however, and could detect serum samples positive to the Indiana serotype of VSV that had SN titers of greater than or equal to 480. Nonspecific reactions were due to cross-reactive group-specific viral proteins that are shared by both serotypes. The cross-reactivity allows the use of a single rapid test in identifying both serotypes of VSV from the other exotic vesicular diseases, especially foot-and-mouth disease. The ELISA titers of serum samples positive for VSV-NJ were comparable with the corresponding SN titers of each sample. The sensitivity, rapidity, and ease of the ELISA system and the use of a single test in identifying both serotypes of VSV from the other exotic vesicular diseases make this ELISA suitable as a rapid diagnostic assay for VS.     

Development of an enzyme-linked immunosorbent assay for Bordetella avium

A Bordetella avium enzyme-linked immunosorbent assay (ELISA) was developed to detect serum antibodies in 1-day-old poults, experimentally infected turkeys, and naturally infected turkeys. The optimized procedure included use of a suspension of whole bacteria coated onto plastic microtiter plates, a 1:200 serum dilution, a 1:3200 dilution of commercially available goat anti-turkey IgG (heavy and light chain) conjugated with horseradish peroxidase, and 0.04% orthophenylenediamine as substrate. A sample/negative (S/N) ratio method of analysis was used to estimate antibody titer from absorbance values. The regression equation used to estimate antibody titers was derived from the testing of naturally infected turkey sera. The equation was derived by plotting the log10 titer of the sera against the S/N ratio at a 1:200 serum dilution. The ELISA was an effective method for detecting antibody to B. avium, and the procedure should prove useful for laboratories equipped for high-volume ELISA testing.     

Comparison of serological tests for the measurement of the primary immune response to avian infectious bronchitis virus vaccines

The immune response of individual chickens exposed in an aerosol apparatus to live commercial avian infectious bronchitis virus (IBV) vaccines was measured by serum neutralization (SN), haemagglutination inhibition (HI), complement fixation (CF) and agar gel precipitin (AGP) tests over a period of 14 weeks.The SN titres showed considerable variation for individual chickens and in a large number of birds were negative until 14 weeks after infection.Positive HI titres were recorded for most birds at one week after infection and persisted throughout the observation period. Some relationship was seen between HI and SN titres particularly in birds showing a high immune response.The results obtained with the AGP were transient, variable and did not compare well with results obtained by the other tests. The highest number of positive AGP reactors were seen two to three weeks after infection.Most birds showed positive titres by the CF test at some time after infection but titres were always low and did not correlate with results obtained by the other tests.Twenty-two weeks after vaccination 23 chickens were challenged by aerosol exposure to the Massachusetts 41 strain of IBV and four days later the trachea, kidneys and oviducts were removed from each bird for attempted virus isolation. Virus was recovered from only one kidney and 11 trachea samples. The mean pre-challenge HI and SN titres of birds from which no virus was recovered were significantly higher than the mash titres of vaccinated birds from which virus was isolated after challenge.