Correlation of enzyme-linked immunosorbent assay titers with protection against infectious bursal disease virus.

We examined the utility of baculovirus-expressed infectious bursal disease virus (IBDV) proteins to act as antigens in the enzyme-linked immunosorbent assay (ELISA). The three IBDV protein antigens tested included 1) a truncated VP2, 2) whole VP2, and 3) the polyprotein products VP2, VP3, and VP4. Serum samples from 2-wk-old commercially reared broilers were collected and tested in the three ELISAs. Serum samples were obtained from 34 different commercial broiler flocks. An average of 14 serum samples (range = 11-17) were tested for each flock. The ELISA results were compared with the percentage of protection of these birds following challenge with IBDV. Fifty 2-wk-old chicks from each of the 34 broiler flocks were challenged with STC classic virus or Del-E variant virus. At 7 days postchallenge, the bursa from each of the birds was removed and bursa/body weights were recorded. Percentage of protection was determined by the number of birds in each challenge group that had normal relative bursal weights compared with unchallenged controls. No evidence was found of a relationship between ELISA data generated with the polyprotein antigen (VP2, VP3, VP4) and percentage of protection observed in the STC and Del-E challenged birds. A significant relationship was found between ELISA data and percentage of protection to STC and Del-E when the truncated VP2 or whole VP2 antigens were used in the ELISA. The results of this study indicate that predicting the percentage of protection against classic or variant IBDV strains in broilers from vaccinated breeder flocks can be improved when VP2 is used as the only antigen in the ELISA.     

An enzyme-linked immunosorbent assay (ELISA) for infectious bursal disease virus.

The application of the indirect enzyme-linked immunosorbent assay (ELISA) for detection of infectious bursal disease virus antibodies in chicken serum was investigated. The test procedure involved the coating of concentrated infectious bursal disease virus antigen onto polystyrene tubes, followed by the addition of chicken anti-infectious bursal disease virus serum and horseradish peroxidase labeled rabbit anti-chicken globulin. As an indicator substrate, 5-aminosalicylic acid, with the oxidant H2O2 was added. The reaction was stopped by 3M NaOH and the colour intensity of the reaction mixtures read in a spectrophotometer at 449 nm. The ELISA test was found to be a precise, sensitive and reproducible means of measuring infectious bursal disease virus antibodies in chicken and turkey sera.     

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.     

Rapid serological profiling by enzyme-linked immunosorbent assay. II. Comparison of computational methods for measuring antibody titer in a single serum dilution

An enzyme-linked immunosorbent assay (ELISA) was used to measure specific antibody activity from a single serum dilution in sera of chickens exposed to Newcastle disease virus (NDV). Observed endpoint titers were used to formulate regression equations, and then absorbance data obtained at a single serum dilution were converted directly to antibody titer by three methods: a correction factor method, a subtraction method, and a double-regression method. Each method was evaluated for three criteria: the overall stability of between-test antibody titer for control sera, the linearity of the relationship of the absorbance values at a single working dilution to the observed antibody titers, and the method's accuracy in predicting titers. Although a nearly linear relationship was obtained for all treatment methods examined, the double-regression method provided the best reduction of between-test titer variation and also best predicted titers.     

An indirect enzyme-linked immunosorbent assay (ELISA) for measuring antibodies in chickens infected with infectious bursal disease virus

An immuno-enzyme assay for measuring infectious bursal disease antibodies in chickens is described. The test is performed rapidly after coating plates overnight with partially purified antigen prepared in cell culture. Coated plates can be stored for at last 4 months. The chromatographically purified rabbit anti-chicken immunoglobulin-G, conjugated to horseradish peroxidase, was used optimally at a dilution of 1:3,000. It could be stored for at least 10 months without a reduction in titer. The test is safe, highly reproducible, specific, and sensitive. Results can be read visually or by spectrophotometry. Antibodies could be detected as early as 4 days postinfection. Serum titers rose rapidly to high levels, ranging from 1:1,600 to 1:25,600 by one week postinfection. High titers persisted for up to one year. The results of this assay compare favorably with results obtained with the agar-gel precipitin and virus-neutralization tests.     

鸡传染性法氏囊病病毒的快速检测

随着 IBDV变异株的出现 ,发病鸡常不产生法氏囊的肉眼病变 ,由于传染性和非传染性因素亦可引起淋巴细胞减少和法氏囊坏死 ,所以 ,采取组织病理学方法诊断 IBDV感染并不完全可靠。由于主动性抗体应答需要一定的时间及大多数雏鸡都带有母源抗体 ,因此血清学早期诊断也不完全可靠。该试验采用本所研究的 IBD快速试纸与经典的琼扩试验 ,在对 IBDV的检测效果上进行了详细的比较 ,介绍如下。1　材料与方法1 .1　 IBD快速检测试纸　由河南省农业科学院生物技术研究所制备提供。1 .2　试验鸡　 1日龄试验鸡 30 0只由河南农业大学试验鸡场提…     

Subclinical infectious bursal disease in commercial broiler flocks in Saskatchewan.

Five commercial broiler flocks, not vaccinated for infectious bursal disease virus, derived from infectious bursal disease virus-vaccinated breeder flocks were surveyed for evidence of bursal damage and infectious bursal disease virus infection. They were compared with two groups of birds raised in isolation. Serum samples from one day old chicks contained maternal anti-infectious bursal disease virus antibodies which declined to undetectable levels by four weeks of age. Serum antibody levels remained undetectable in both control groups and one commercial flock, whereas four of the five commercial flocks had actively produced anti-infectious bursal disease virus antibodies by slaughter age. The weight of bursae from infectious bursal disease virus-positive flocks declined as compared to controls after four weeks of age. The decline in weight correlated with the appearance of histopathological lesions. Infectious bursal disease virus antigen was demonstrated in selected infected bursae and infectious bursal disease bursae and infectious bursal disease virus was isolated from some of these damaged bursae. Clinical infectious bursal disease was not observed in any of the commercial flocks. The importance of subclinical bursal damage and immunosuppression is discussed.     

Fowl cholera immunity in broiler breeder chickens determined by the enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies in broiler breeders vaccinated (wing web) with the CU fowl cholera vaccine. Birds were bled weekly from 9 to 26 weeks, every other week from 26 to 40 weeks, and every 4 weeks from 40 to 56 weeks of age. Overall mean ELISA antibody titers (9 to 56 weeks) reported as log10 values and survivability of the vaccinates after virulent challenge were as follows: unvaccinated--5.75, 48%; birds vaccinated once at 8 weeks--5.91, 78%; birds vaccinated twice at 8 and 14 weeks--6.11, 100%; birds vaccinated thrice at 8, 14, and 20 weeks--6.23, 100%; birds vaccinated twice at 8 and 20 weeks--6.12, 100%; and birds vaccinated twice at 8 and 20 weeks (plus fowl pox at 8 weeks)--6.08, 95%. Survivability in the vaccinates after virulent challenge with strain X-73 Pasteurella multocida was 100% in birds with ELISA antibody titers (log10) greater than 6.02. Under the conditions of this experiment, birds vaccinated two or three times between 8 and 20 weeks proved to be sufficiently immune at 56 weeks of age to withstand a virulent fowl cholera challenge. Birds not vaccinated or vaccinated only once at 8 weeks were not sufficiently immunized to withstand virulent challenge.     

Subclinical infectious bursal disease in an integrated broiler production operation.

A field study was designed to determine the prevalence of subclinical infectious bursal disease (IBD) in broiler chickens from a commercial poultry company. Bursae of Fabricius (BF) from two vaccinated and three nonvaccinated broiler flocks were evaluated histologically, and antibody profiles of these broiler and matched parent breeder flocks were established. Lesions of IBD, including lymphoid necrosis, stromal edema, and infiltrates of heterophils and macrophages, were first detected in BF at 24 days of age in both vaccinated and nonvaccinated chickens. At 41 days, all BF had lesions characteristic of IBD, including severe lymphoid depletion, proliferation of epithelial cells, and mild fibroplasia. Although mean maternal antibody levels (measured by enzyme-linked immunosorbent assay) in broilers were apparently protective through day 12, IBD antibodies decreased to nonprotective levels (below 1,000) by day 16 or 20. Titers began to increase by day 28 or 32 because of field exposure. Sentinel birds, placed with broiler flocks, also developed IBD antibody titers. Broiler breeders had low and nonuniform antibody titers. Prevalence of field IBD exposure was high, and existing vaccination programs were not effective.     

Development of an enzyme-linked immunosorbent assay for detecting infectious bursal disease virus (IBDV) infection based on the VP3 structural protein

Infectious bursal disease virus (IBDV) causes a contagious immunosuppressive disease in chickens. The aim of the present study is to develop an enzyme-linked immunosorbent assay (ELISA) using the expressed VP2 or VP3 protein of IBDV as the coating antigen for detecting antibodies to IBDV. Experimental results were compared with virus neutralization assay and a commercial-available ELISA. These assays were used to examine the sera from farm chickens and chickens vaccinated experimentally. The VP3-based ELISA had a higher correlation coefficient (R(2)) of 0.812 with a commercial ELISA kit at a serum dilution of 1:500 than that of VP2-based ELISA (R(2)) of 0.671. The relative sensitivity between virus neutralization and VP2-ELISA and VP3-ELISA was 96% (251/262) and 100% (262/262), respectively, and that between virus neutralization and a commercial ELISA was 99% (257/261). Additionally, compared with virus neutralization assay, the reference technique for diagnosing IBDV, VP3-based ELISA had an agreement value of 99%, superior to that of VP2-based ELISA (95%) or the commercial kit (89%). These results revealed that the capability of either VP2-ELISA or VP3-ELISA in detecting the field chicken sera was comparable to the commercial one, which is generally used to replace the virus neutralization assay. However, the preparation of VP3 is derived from an Escherichia coli expression system with a high yield and purification efficiency by Ni(2+)-NTA gels, which is more favorable to the insect cell-derived particles formed by VP2. Therefore, VP3-ELISA could be developed as an efficient and low cost diagnostic method for IBDV infection in field chickens.     

Rapid detection of viral-specific antibodies by enzyme-linked immunosorbent assay (ELISA)

The development of three separate rapid ELISAs for detecting antibodies in host serum to three different viruses is described. These include: 1. A direct antigen assay using enzyme labelled anti-canine Ig for detecting antibodies to canine parvovirus, 2. A competitive ELISA using a feline infectious peritonitis virus-specific monoclonal antibody labelled with enzyme, and 3. A competitive ELISA using an equine infectious anemia virus-specific monoclonal antibody and enzyme labelled antigen, p. 26. The utility and benefits of each of the three approaches is emphasized.     

Detection and quantification of antibodies to infectious bronchitis virus by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) for detecting and quantifying antibodies to infectious bronchitis virus (IBV) is described. Purified antigen, prepared on sucrose density gradients, was required to decrease the nonspecific background, and saline was found to be superior to bicarbonate buffer for coating the cuvettes with antigen. The sensitivity of the test in measuring antiserum titers could be altered greatly and linearly by adjusting the protein content of the antigen. The ELISA was able to detect an antibody response to IBV infection earlier than the virus-neutralization (VN) test. Antibody titers obtained by ELISA were considerably higher than those obtained by VN. Serotypes of IBV could not be differentiated with ELISA because of extensive antiserum cross-reactivity. The utility of ELISA in studies on IBV is discussed.     

Standardization and application of the enzyme-linked immunosorbent assay for infectious bronchitis

The indirect enzyme-linked immunosorbent assay (ELISA) was used to detect antibody to infectious bronchitis virus in chickens. The serum-neutralization test for infectious bronchitis (SNIB) was used as a reference serologic test. The ELISA proved to be useful for monitoring antibody responses following vaccination of leghorn chicks. The titers obtained with the ELISA and SNIB showed an overall correlation, but results suggest the involvement of different antibody classes.     

Comparison of filter-paper-eluted whole blood with serum in fowl cholera serology using the enzyme-linked immunosorbent assay

Two methods for collecting blood for measuring antibody activity of Pasteurella multocida were compared. Whole blood was collected on filter-paper strips, dried for 48 hr at room temperature, and then stored in sealed plastic bags at 4 C. Blood was also collected in the usual manner with a needle and syringe, and serum was harvested and stored at -20 C until tested. Eluates of whole blood, obtained by overnight elution of two 4.8-mm discs in 200 microliters of buffered saline at 4 C, were compared with conventionally harvested serum for antibody activity by enzyme-linked immunosorbent assay (ELISA). Paired samples, taken from the same bird at the same time, showed no significant difference (P less than 0.05) in antibody activity as measured by absorbance when the disc-elution process itself was considered to be a 1:20 dilution. It was concluded that eluates of blood, derived from whole blood dried on filter-paper strips, may be used as an alternative to sera in ELISA for measuring P. multocida antibody activity.     

用改良的RT-PCR技术快速检测传染性法氏囊病病毒

本研究将最新的病毒核酸纯化技术和单管ＲＴ－ＰＣＲ技术相结合,应用于ＩＢＤＶ的诊断研究,对４０余个病毒样品进行扩增反应,均取得令人满意的效果。该技术灵敏快速,从核酸纯化到电脉检测ＰＣＲ产物只需５～６ｈ,反应灵敏度比传统方法至少提高１００倍。所设计的引物对毒株的适用范围较广,对１２个ＩＢＤＶ毒株和１２个病变法氏囊样品均得到分子量一致并与设计相符的扩增产物     

Fowl cholera immunity induced by various vaccines in broiler minibreeder chickens determined by enzyme-linked immunosorbent assay

Broiler minibreeder hens were vaccinated for protection against fowl cholera at 12 and 21 weeks of age using several vaccination schemes, which included a live Pasteurella multocida (CU strain) vaccine, two commercial polyvalent fowl cholera oil-based bacterins, and two experimentally prepared polyvalent oil-based bacterins. Some treatment groups received only live or killed vaccines, whereas others received a live vaccine at 12 weeks followed by a killed product at 21 weeks. At 42 weeks of age, all birds that received the live CU vaccine twice or once followed by a bacterin survived challenge. Birds that received killed vaccines only were significantly less protected but still showed a respectable survival rate of 86%. All unvaccinated controls died within 72 hr after challenge. At 72 weeks of age, overall protection was lower than that at 42 weeks, regardless of vaccination treatment. Antibody titers were usually higher in birds that received bacterins than in those receiving live vaccines, yet overall protection was still greater in those birds that received the live cholera vaccine twice.     

肉仔鸡传染性法氏囊病并发大肠杆菌病的诊治

传染性法氏囊病是由鸡传染性法氏囊病病毒引起的一种急性、高度接触性传染病，是目前危害养鸡业的主要传染病之一。该病的发生可造成机体免疫力下降，产生长期的、严重的免疫抑制现象，降低机体对各种疾病的抵抗力，使鸡容易感染其它传染病，从而给一些养殖场或养鸡户造成巨大的经济损失，笔者在２００２年２月２８日遇到一典型病例，现报道如下：１流行病学调查山东省临沭县青云镇韩村村民王怀顺饲养的３０００只AA肉仔鸡，采用地面平养，饲养密度为２５只／m２。免疫程序为：７日龄使用新支二联苗饮水，１４日龄法氏囊疫苗饮水，２８日龄…