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1.
An enzyme-linked immunosorbent assay using the expressed protein sigma B as the coating antigen (sigma B-ELISA) for detecting antibody to avian reovirus (ARV) in chickens was developed and compared with a conventional ELISA. Both ELISA s and a serum neutralisation (SN) test were used to test the sera from experimentally vaccinated and farm chickens. The sigma B-ELISA could clearly distinguish the SN-positive and -negative sera in 38-week-old chickens. The correlation rate between SN and a sigma B-ELISA was 100 per cent (65/65), and that between SN and conventional ELISA was 84 per cent (55/65). With the sigma B-ELISA, all SN-negative sera had low absorbance values (below 0.06), and the absorbance values correlated closely with the SN titres. However, the sera which were antibody-negative by SN had various absorbance values, ranging from 0.07 to 0.39 in the conventional ELISA. Hence, the sigma B-ELISA had lower non-specific binding reactions than the conventional ELISA against sera from ARV -negative birds. Antibody against ARV could be detected by sigma B-ELISA after vaccination. Absorbance values peaked 4 weeks after vaccination at 2 weeks of age and were maintained until the birds were 27 weeks old. The results suggest that the presence of antibody against viral protein sigma B in birds may be used as a good indicator by the sigma B - ELISA for detecting immune status of a chicken flock or to detect chickens infected with ARV.  相似文献   

2.
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.  相似文献   

3.
An ELISA was developed using staphylococcal protein A linked with horseradish peroxidase for detecting IgG antibody of rabies virus in human and carnivore sera (80 human, 270 fox, 40 cat, 35 marten, 5 badger and 4 polecat sera were tested in the present work). In comparison with the serum neutralization (SN) test in cell culture, close overall agreement was obtained particularly in human and cat sera (97.5%). Two post-vaccination human sera were found positive with ELISA values of 2.16 and 2.65 IU/0.2 ml, but with SN titers less than 1:10. All prevaccination human sera were found negative by both tests. Regression analysis on 30 post-vaccination human sera revealed better correlation between ELISA and SN test at a serum dilution of 1:100 than at lower serum dilutions of 1:80 or 1:20. The correlation coefficient (r) was 0.73 (p less than or equal to 0.0001).  相似文献   

4.
应用酶联免疫吸附试验（ＥＬＩＳＡ）、琼脂扩散沉淀试验（ＡＧＰ）和鸡气管环培养中和试验（ＳＮｉｎＴＯＣｓ）三种常规血清学方法对实验鸡血样的鸡传染性支气管炎病毒抗体进行了检测。从实验鸡血样的检测结果表明，ＥＬＩＳＡ和气管环中和试验的灵敏度较好，而ＡＧＰ的灵敏度相对较差，但三者均有较好的特异性。对田间送检血样的检测结果表明，ＥＬＩＳＡ与气管环中和试验、ＥＬＩＳＡ与ＡＧＰ的一致性均较好，而气管环中和试验与ＡＧＰ的一致性较差。ＥＬＩＳＡ效价与气管环中和试验效价的相关系数为０．８４  相似文献   

5.
Specific-pathogen-free chickens were infected via the trachea when 4 weeks old with 2000 plaque-forming units (PFU) of the virulent Australian infectious laryngotracheitis (ILT) virus strain CSW-1. Titers of ILT virus in the trachea were greatest (10(7.0) PFU/ml in washings, 10(6.0) PFU/g of tissue) 2-4 days postinfection (PI). Infectivity then declined rapidly, to become undetectable by 7 days PI, although highly localized areas of ILT antigen in the tracheal epithelium were occasionally observed by fluorescent antibody staining at 7 and 8 days PI. Tracheal organ cultures established 7 and 8 days PI provided no evidence of latent ILT virus infection at this immediate post-acute stage of pathogenesis. ILT virus was not isolated from peripheral blood leukocytes or lymphoid organs (spleen, bursa, thymus). ILT virus was found in the trigeminal ganglia and/or brain in 14 of 36 chickens (40%) examined between 4 and 7 days after intratracheal inoculation, but it was not in these tissues in five chickens examined at 8 days PI. Virus was also detected at 6 days PI in the trigeminal ganglia in one of five chickens infected by the conjunctival route. These data indicate that the early pathogenesis of ILT (CSW-1) infection frequently involves the tissues of the nervous system. In acute ILT in 4-week-old chickens, interferon-alpha/beta activity was not detectable in serum or tracheal exudates within 14 days PI, but tracheal washings contained significant virus-neutralizing activity by 7 and 8 days PI. In 3-day-old chickens infected via the trachea with 200 PFU of ILT CSW-1, the clearance of ILT virus from the trachea was similar to that observed in 4-week-old chickens, but ILT virus spread systemically to the livers of 20% by 5-7 days PI.  相似文献   

6.
A microtitre ELISA has been established for the quantitation of antibodies to bovine viral diarrhoea virus (BVDV). Single dilutions of sera were assayed and units of antibody were calculated from a standard curve. In order to detect the maximum number of responding animals both IgG1 and IgG2 antibody should be assayed, although detection of IgG1 alone was nearly as effective. The ELISA was as sensitive as the virus neutralization test for detection of antibody; comparison of an ELISA that detected IgG1 plus IgG2 antibody to BVDV with the virus neutralization test gave a correlation coefficient (r) of 0.89 (P less than 0.001 for 95 compared sera). Although similar amounts of IgG1 and IgG2 antibodies were present in sera from both experimentally- and naturally-infected cattle, antibody to BVDV in colostrum and in the sera from young calves was predominantly IgG1. The number of adult cows with antibody was 40 out of 41 while 36 of 44 calves reared in a beef unit were found to have produced antibody by the time they were 31.5 weeks old, an indication of the high prevalence of BVDV in the cattle population.  相似文献   

7.
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.  相似文献   

8.
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.  相似文献   

9.
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.  相似文献   

10.
Vaccination and challenge experiments using infectious bronchitis virus (IBV) were conducted on groups of specific-pathogen-free chickens. Three weeks post-vaccination with one of the four IBV strains used, chickens were challenged with the homologous immunizing strain of IBV. Subsequently, the chickens were sacrificed, their tracheas were examined for ciliostasis, and the specific IBV antibody content of their sera was measured by enzyme-linked immunosorbent assay (ELISA). Results showed that protection was conferred by primary vaccination, as ciliostasis was not observed in tracheas from groups vaccinated and then challenged. No protection was observed in control groups that received only a challenge exposure, and the virus was readily recovered from their tracheas. Homologous protection was present in chickens that had ELISA titers as low as 624 and neutralization indices as low as 2.9, whereas susceptible controls had titers of less than 100 and less than 1.0, respectively.  相似文献   

11.
The immunoglobulin response of chickens to colonization by Campylobacter jejuni isolates B-540 and Clin-1 was monitored. Chicken humoral IgG and biliary secretory IgA (sIgA) responses were assessed by enzyme-linked immunosorbent assay (ELISA). Samples were taken from 128 C. jejuni-colonized chickens and 104 uncolonized chickens housed in a controlled environment. An indirect ELISA was performed using the homologous isolate of C. jejuni as the capture antigen and was developed with the specific goat anti-chicken IgG or IgA alkaline phosphatase conjugates. The ELISA absorbance values of the test samples at 405 nm (serum diluted 1:32 and bile diluted 1:10) were normalized in direct proportion to standard sera and bile sample values. In the colonized chickens, humoral IgG activities were highest at hatch, dropped to their lowest level after 2 weeks, and increased by 8 weeks to levels similar to those detected at hatch. The sIgA activity was lowest at hatch and increased by 4 weeks in colonized chickens while remaining lower in the control chickens. Chickens colonized with isolate B-540 showed a primary sIgA response during the first 4 weeks and reached a plateau over the final 4 weeks. In spite of these limited humoral and secretory immunoglobulin responses, once the chicken ceca was colonized by C. jejuni, the organism persisted throughout the 8-week experiment.  相似文献   

12.
An indirect ELISA has been developed to detect Salmonella typhimurium antibodies in chicken sera, using whole bacterial cell protein, flagellar protein or lipopolysaccharide as antigens. In experimental infections high concentrations of S typhimurium-specific IgG persisted after the faecal excretion of S typhimurium had ceased, whereas the specific IgM response was transitory. Some uninfected chickens placed in contact with experimentally infected birds developed high IgG titres in the absence of detectable faecal excretion. Other S typhimurium strains, which varied in their invasive abilities, also induced high titres of IgG. The ELISA allowed chickens infected experimentally with S typhimurium to be differentiated from chickens infected with 10 other serotypes, including S enteritidis. The use of whole blood in place of serum in the ELISA reduced the titres slightly. The storage of serum dried on to filter paper strips for four weeks produced little change in ELISA antibody titre, and the treatment of such strips with phenol or chloroform vapour had little or no effect on the antibody titre.  相似文献   

13.
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.  相似文献   

14.
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.  相似文献   

15.
Chickens highly susceptible to avian leukosis virus (ALV) infection and tumors, with and without ALV subgroup A maternal antibody (MAB), were infected with a field strain of ALV subgroup A at hatching. Viremia, antibody development, cloacal and albumen shedding, and tumors in chickens with MAB (MAB+) were compared with those in chickens lacking MAB (MAB-). At 18 weeks of age, the incidence of viremia was significantly lower in MAB+ chickens than in MAB- chickens; further, MAB significantly reduced the proportion of tolerantly infected (viremic antibody-negative) chickens. Cloacal shedding of ALV at 22 weeks of age and shedding of ALV group-specific (gs) antigen in albumen of eggs from all laying hens at 30-32 weeks of age were significantly lower in MAB+ hens than in MAB- hens. The incidence of ALV-induced tumors was lower in MAB+ chickens than in MAB- chickens, significantly so in one of three trials conducted. These results suggest that MAB may influence the development of viremia, antibody, and shedding of ALV following massive exposure to virus at hatching.  相似文献   

16.
A total of 291 eight-week-old chickens were exposed to chickens infected with either of two Australian lentogenic strains (V4 and AVL NDV-1) of Newcastle disease virus (NDV). At 3 weeks after exposure, all chickens exposed to V4 infected chickens had developed haemagglutination-inhibition (HI) antibody. All chickens exposed to AVL NDV-1 virus infected chickens had developed HI antibody 5 weeks later. This sudden late appearance of HI antibody, to titres higher than those observed with V4 chickens, was explained by V4 virus being introduced to the AVL NDV-1 group of chickens. When groups of these chickens were challenged with Roakin virus (mesogenic NDV) at 3 weeks and Fontana 1083 virus (viscerotropic velogenic NDV) and Texas GB virus (neutrotropic NDV) at 3, 5, 10 and 21 weeks only three chickens developed clinical illness one of which died. These chickens were one AVL NDV-1 chicken contact challenged with Fontana 1083 virus at 3 weeks, one V4 chicken oronasally challenged with Texas GB virus at 5 weeks and one V4 chicken challenged oronasally with Fontana 1083 virus at 10 weeks. Susceptible non-vaccinated chickens died soon after challenge. Challenge by oronasal infection with 10(7.0) ELD50 of virus or contact with susceptible infected chickens enabled virulent virus to be isolated from most chickens and was accompanied by a large anamnestic increase in serum HI antibody.  相似文献   

17.
The B1 strain of Newcastle disease virus (NDV-B1), which is nonpathogenic for newly hatched chickens, killed embryos when it was used to inoculate chicken eggs at embryonation day 18. Treatment of NDV-B1 with an alkylating agent, ethylmethane sulfonate (EMS) markedly reduced the pathogenicity of the virus for 18-day-old chicken embryos. Eggs inoculated with the modified virus (NDV-B1-EMS) hatched, and the virus was isolated from lungs and spleen of 1-day-old chickens. The hatched chickens developed antibody to NDV and were protected against challenge exposure (at 4 weeks of age) with a highly virulent GB-Texas strain of NDV. Presence of maternal antibody to NDV in embryonating eggs did not influence the protective ability of NDV-B1-EMS, which also induced protective immunity when administered to 4-week-old chickens. The 50% protective dose of NDV-B1-EMS in maternal antibody-negative and -positive embryos was calculated to be 10.77 and 17.70 embryo 50% lethal doses, respectively. Results of the study indicated that NDV-B1-EMS may be used as an embryo vaccine to protect chickens against Newcastle disease.  相似文献   

18.
Three glycoproteins of infectious laryngotracheitis virus (ILTV), gC, gE, and gp60, were expressed in Escherichia coli as fusion proteins with a 6-histidine tag at their amino termini. The proteins expressed, designated as r-gC, r-gp60, and r-gE, all retain their antigenicity, as revealed by Western blot with chicken antiserum against ILTV. However, only r-gp60 and r-gE, but not r-gC, were found to be soluble. The soluble r-gp60 and r-gE were purified by a nickel column and then used as the enzyme-linked immunosorbent assay (ELISA) antigen for detecting ILTV-specific antibodies. The diagnostic potential of r-gE and r-gp60 ELISA was assessed with the use of sera prepared from vaccinated or unvaccinated chickens of either specific-pathogen-free (SPF) or field origins. The result shows that r-gp60 and r-gE ELISA could discriminate vaccinated SPF chickens from unvaccinated ones 2 wk postvaccination. Moreover, r-gp60 and r-gE ELISA could also discriminate vaccinated field flocks from unvaccinated ones. This result indicates that r-gp60 and r-gE might serve as an alternative ELISA antigen for detecting ILTV-specific antibodies. Moreover, r-gp60 or r-gE ELISA might play an important role in the eradication of infectious laryngotracheitis (ILT) in the future when the gp60- or gE-deleted marker vaccine of ILT is available.  相似文献   

19.
To examine the specificity of the antibody response to the influenza hemagglutinin and the generation of antigenic variants, chickens were immunized against the highly virulent H5 virus A/Ty/Ont/7732/66 (H5N9) and then challenged with a lethal dose of the virus. The antibody responses of these chickens to the hemagglutinin (HA) were examined with an enzyme-linked immunosorbent assay (ELISA) in which their sera were titrated for the ability to block the binding of monoclonal antibodies (MAbs) to five distinct neutralizing epitopes on the viral HA. Based on the ELISA results, a majority (5/6) of the chickens produced antibodies to three of the five neutralizing epitopes on the viral HA. After challenge, two of six immunized chickens shed virus and died; antigenic comparisons of isolates from these two chickens indicated the presence of an antigenic variant; i.e., there was a change in one neutralizing epitope on the HA of virus shed by one chicken. None of the chickens had produced antibodies to this particular epitope on the viral HA. Inoculation of chickens with this variant resulted in 100% mortality, demonstrating that a change in this particular epitope did not alter the virulence of the virus. These studies indicate that chickens immunized against highly virulent influenza viruses may excrete virulent variants following challenge with live virus.  相似文献   

20.
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.  相似文献   

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