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1.
A dot-immunobinding assay, amplified with avidin and biotin (DAB assay), was used to detect serum antibodies to Mycoplasma iowae in immunized turkeys. The DAB assay was used to test serum samples from 122 commercial market turkey flocks obtained from four Iowa processing plants. The samples were pooled and tested for the presence of antibodies to four species of Mycoplasma spp. considered to be important pathogens for turkeys: M. gallisepticum (MG), M. iowae (MI), M. meleagridis (MM), and M. synoviae (MS). The occurrence of antibodies against these mycoplasmas, as determined by the DAB assay, were 5.7% for MG, 18.0% for MI, 77.9% for MM, and 9.8% for MS.  相似文献   

2.
The sensitivity and specificity of the indirect micro-enzyme-linked immunosorbent assay (ELISA) was compared with that of the rapid serum-plate test (RSPT) and the hemagglutination-inhibition test (HIT) in detecting antibodies to Mycoplasma gallisepticum (MG) and M. synoviae (MS). Membrane antigens of MG strain S6 and MS strain NEL 61800 were used. ELISA was performed with single MS and single MG antigens and a combined MS/MG antigen. The MS-ELISA was as sensitive as the MS-RSPT and more sensitive than and as specific as the MS-HIT in detecting antibodies to MS. The MG-ELISA was less sensitive than the MG-RSPT and slightly less sensitive than the MG-HIT in detecting antibodies to MG in chickens experimentally infected with MG R strain but more sensitive in detecting antibodies in chickens infected with MG F strain. MG-ELISA resulted in fewer cross-reactions than the MG-RSPT but more than the MG-HIT. The combined MG/MS-ELISA was as sensitive as the ELISA with its individual antigen components. No nonspecific reactions were observed with sera from MG/MS-free flocks. The combined MG/MS-ELISA was found to be a practical screening test for antibodies to both MS and MG. Further improvement of the sensitivity and the specificity of the MG antigen is desirable.  相似文献   

3.
Egg yolk was evaluated in the enzyme-linked immunosorbent assay (ELISA) as an alternative source of antibodies for detection of Mycoplasma gallisepticum (MG) and M. synoviae (MS) infections in chickens. There was no statistically significant difference (P greater than 0.05) between the ELISA geometric mean titers (GMTs) of saline-diluted egg yolk and chloroform-extracted egg yolk, and both preparations had a high correlation coefficient (0.87 for MG; 0.97 for MS). The saline-diluted and chloroform-extracted yolk had a relative sensitivity of 90% and specificity of 98% in the MG ELISA; in MS ELISA they were 100% and 96%, respectively. Hemagglutination-inhibition (HI) results with chloroform-extracted samples were satisfactory, but those with saline-diluted samples were not. Neither preparation was satisfactory for use in the rapid plate agglutination (RPA) test. A 1-ml sample of yolk was compared with the whole-yolk method. The chloroform-extracted whole yolk yielded a significantly higher (P less than 0.05) GMT in the MG ELISA; however, there was no statistically significant difference (P greater than 0.05) between GMTs yielded by the two procedures in the MS ELISA. The correlation coefficients for the two sampling methods were 0.73 for MG ELISA and 0.63 for MS ELISA. ELISA detected no statistically significant difference (P greater than 0.05) between GMTs of serum and chloroform-extracted yolk from individual birds. Results with the HI test were comparable to those with ELISA on the same samples. The RPA test yielded comparable results on the serum samples. No statistically significant differences (P greater than 0.05) were observed in HI or ELISA antibody levels between egg-yolk samples and sera on random samples collected from nine flocks that were MG- and MS-free or were infected with MG, MS, or both; however, egg-yolk samples tended to have slightly higher titers than sera in both tests. The optimum screening dilution of chloroform-extracted yolk for detecting MG and MS antibodies by ELISA was 1:800.  相似文献   

4.
检测禽流感病毒抗体的重组核蛋白间接ELISA方法的建立   总被引：9，自引：3，他引：9  
以大肠杆菌系统表达的H9N2亚型禽流感病毒（AIV）核蛋白（NP）为抗原，建立了禽流感间接酶联免疫吸附试验抗体检测技术（NP—ELISA）。对263份待检血清（包括临床收集的243份血清和20份H9N2亚型AIV免疫鸡阳性血清）进行检测，NP—ELISA与琼脂免疫扩散试验（AGP）的总符合率为83．3％，与血凝抑制试验（HI）的总符合率为92％。特异性试验表明，NP—ELISA方法可以检测H5、H7和H9亚型AIV特异性抗体，检测为阳性的血清样品能够被阳性鸡胚尿囊液阻断。敏感性试验证实，NP—ELISA最早可以检测鸡感染后7d的血清样品，并于感染后10d确定100％血清阳性，而AGP检测直到首免后21～28d才出现部分血清阳性，HI检测直到10～14d才出现部分血清阳性，并且NP-ELISA要比HI敏感4～40倍。试验证明，NP—ELISA是检测AIV血清型特异性抗体的一种特异、敏感、快速、经济的血清学检测技术。  相似文献   

5.
Selected immunogenic proteins of Mycoplasma gallisepticum (MG) strain R and M. synoviae (MS) isolate F10-2AS were purified from sodium dodecyl sulfate-polyacrylamide gels. Purified MG proteins of 65 to 63 (p64) kilodaltons (kDa), and 26 and 24 (p26/24) kDa, and purified MS proteins of 53 (p53) kDa, 41 (p41) kDa, and 22 (p22) kDa were evaluated as potential antigens for an enzyme-linked immunosorbent assay (ELISA). Chicken antisera to MG, MS, or oil-emulsion vaccines were used to evaluate these purified proteins as antigens in a dot-ELISA. MG antigen p64 detected antibodies 3 days after the serum plate agglutination (SPA) test and 7 days before the hemagglutination-inhibition (HI) test. Antigen p64 detected antibodies to 12 MG isolates, and in sera from field outbreaks of MG. No cross-reactions with MS-positive antisera were seen with antigen p64. MG antigen p26/24 did not perform as well as p64. MS antigen p41 detected antibodies 5 days after the SPA test and at least 11 days before the HI test, and in sera from field outbreaks of MS. However, some MG-positive antisera reacted with p41. MS antigens p53 and p22 did not perform well.  相似文献   

6.
Three experimental strains of breeder chickens were accidentally exposed to Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), presumably from a newly introduced group of leghorn-type pullets. The experimental strains subsequently became infected and were diagnosed positive for MG and MS by the serum plate agglutination (SPA) test and confirmed by the hemagglutination inhibition (HI) test and the polymerase chain reaction (PCR) of tracheal swabs. Treatment with 10 mg/kg enrofloxacin via drinking water for 14 days was elected. Before and after initiation of treatment, MG and MS were monitored for changes by SPA, HI, PCR, and culture, with sampling intervals ranging from 1 wk to 7 wk. MG and MS SPA, HI, PCR, and culture were performed at each sampling period, with the exception of weeks 1.0 and 6.5. Week 1.0 included SPA and His for MG and MS. Week 6.5 included PCR and culture for MG and MS. The MG and MS SPA results were positive throughout the 29-wk trial period. MG HI titers declined until the last sampling, whereas the MS HI titers did not decline significantly. PCR for MG yielded only one positive result, which occurred before treatment. MS PCR remained positive throughout the trial period. MG was never isolated from any sample; however, one MS organism was isolated during treatment. The treatment regimen was effective for MG on the basis of PCR results. Treatment with enrofloxacin did not eliminate SPA reactions during the 29-wk trial period. MG HI titers remained in the suspicious range throughout the remainder of the trial period. Four weeks after the treatment ended, MG HIs were reduced by approximately 40%, with MS HIs remaining high throughout the 29-wk period. PCR appeared to be a sensitive and specific test on the basis of correlation with HIs. On the basis of the isolation of MS during treatment and continued subsequent PCR positive reactions, the treatment for MS with enrofloxacin was not as efficacious as for MG.  相似文献   

7.
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.  相似文献   

8.
Both Mycoplasma gallisepticum (MG) and M. synoviae (MS) antigens prepared for the routine haemagglutination inhibition (HI) test were diluted and absorbed to the separate pieces of durapore membrane for the measurement of dot-immunobinding (DIB) titers of test sera. Besides, durapore strips bearing both antigens were employed for a DIB test with chicken sera definitely diluted 100-fold. Shortening of reaction time of chicken sera with antigens as well as with the secondary serum markedly eliminated non-specific DIB reactions exhibited at low dilutions although the same condition was not so effective on the elimination of non-specific reactions among rabbit hyperimmune sera. Rapid and specific development of DIB antibody which continued at high titer up to 1:640 for 10 weeks postinoculation was proved in the sera of SPF chickens inoculated with MG or MS, while DIB titers of sera from uninoculated chickens remained 1:20 or lower. Non-specific reactions, which occurred in the routine serum plate agglutination test with a part of sera from the inoculated chickens, were not exhibited in the DIB as well as in the HI test with the same sera. Results of the DIB test with serum samples from 287 conventionally reared chickens definitely diluted 100-fold coincided with the results of HI test at a level of 90% with MG and 89% with MS antigen. This technique seems to be useful for a rapid, simple and specific diagnosis of avian mycoplasmosis.  相似文献   

9.
Agar gel immunodiffusion (AGID) and counter-immunoelectrophoresis (CIEP), complement fixation (CF), radio-immunoassay (RIA), haemagglutination (HA) and haemagglutination inhibition (HI) tests were compared in their efficiency for the detection of bovine rotavirus antigens and antibodies. As a test for antigen using hyperimmune serum, CIEP was found to have advantages over AGID by being more rapid as well as approximately four times more sensitive regardless of whether the antigen was of faecal or tissue culture origin. The CF test was more sensitive than either of the immunodiffusion procedures studied for antigen detection, but was more tedious to perform and of limited use as some faecal samples exhibited anti-complementary activity. For measurement of rotavirus antibody the radio-immunoassay (RIA) was the most sensitive technique and the CIEP least sensitive. Using the RIA a limited survey of cattle demonstrated that approximately 75% of the animals tested possessed specific antibody to rotavirus.  相似文献   

10.
An indirect enzyme-linked immunosorbent assay (ELISA) was developed and tested for its ability to detect humoral response to Mycoplasma gallisepticum in chickens. Two antigens were used in the solid phase of the assay. Antigen 1 was a membrane-derived sodium dodecyl sulfate (SDS)-solubilized preparation; Antigen 2 was prepared in the same manner as Antigen 1 but was passed through an immunoadsorbent column containing rabbit anti-medium antibodies. Test conditions were optimized for incubation times and temperatures. Antigen, serum, and enzyme conjugate concentrations were standardized, and reproducibility was determined. A baseline value, representing a positive or negative result, was established independently for both antigens. The assay was then used to detect anti-M. gallisepticum antibodies in experimentally infected chickens. Serum samples collected at 0, 2, 5, 7, 10, 14, 21, 28, and 35 days postinfection (PI) were analyzed by serum plate agglutination (SPA), hemagglutination-inhibition (HI), and ELISA with both Antigens 1 and 2. ELISA was found to be less sensitive but more specific than SPA and more sensitive than HI. The ELISA was more sensitive with Antigen 1 than with Antigen 2. The former assay correctly identified 79% of the serum samples positive for M. gallisepticum by 7 days PI and 100% of the positive birds by 35 days PI. When the absorbance values for each group of birds were averaged, the ELISA successfully identified the M. gallisepticum-infected birds as uniformly positive 7 through 35 days PI and correctly identified all other groups negative for M. gallisepticum through 35 days PI.  相似文献   

11.
An enzyme-linked immunosorbent assay (MYCO-ELISA) was developed to detect antibodies to Mycoplasma gallisepticum (MG) in chicken sera. The assay was standardized in terms of optimum antigen concentration, serum dilution, conjugate dilution and incubation temperature, and time. The MYCO-ELISA antigen was prepared from MG whole bacterial cell or its disrupted cell suspension. Both preparations showed strong affinity for binding or adsorbing to the surface of polystyrene wells of the microtiter plate. The MYCO-ELISA was more sensitive than the hemagglutination-inhibition test. However, cross-reactions were observed with sera from M. synoviae (MS)-infected birds.  相似文献   

12.
Two commercial enzyme-linked immunosorbent assay (ELISA) kits, seven serum plate agglutination (SPA) antigens, and the hemagglutination-inhibition (HI) test for antibodies to Mycoplasma gallisepticum (MG) were compared for sensitivity and specificity using known MG-positive and MG-negative sera from leghorn chickens. All SPA antigens proved to be highly sensitive when testing MG-positive sera. Laboratory-prepared SPA antigens yielded fewer positive reactions when testing MG-negative sera than commercial SPA antigens. Both MG ELISA kits showed high rates of positive reactions when testing sera from birds given commercial M. synoviae bacterin, fowl coryza (Haemophilus paragallinarum) bacterin, inactivated infectious bursal disease virus vaccine, and to a lesser extent fowl cholera (Pasteurella multocida) bacterin. Immunization with Frey's medium with 12% swine serum-in-oil or Staphylococcus aureus-in-oil resulted in sera which yielded numerous positive ELISA reactions. During the first 1 to 3 weeks, antibodies induced by experimental infection with MG were better detected by the SPA test than by the ELISAs and the HI test, thus confirming the SPA test's importance in Mycoplasma diagnostic serology. The HI test can serve to confirm positive SPA results.  相似文献   

13.
An immunoperoxidase monolayer assay (IPMA) has been developed to detect antibodies against swine influenza A virus (SIV) in pig sera. The test was evaluated by using sequential sera from pigs experimentally infected with H1N1 subtype of SIV. Two hundred field serum samples that had been examined by the hemagglutination inhibition (HI) test were also tested. Antibodies specific to SIV were detected as early as 3 days postinoculation (dpi) in the IPMA test as compared with 7 dpi by the HI test. Unlike HI, no serum treatment was required in the IPMA test. Regardless of the virus used in the test, IPMA detected antibodies to both H1N1 and H3N2 subtypes of SIV whereas HI detects antibodies against either H1N1 or H3N2, depending upon the virus used in the test. Results of this study indicate that IPMA is a useful test for screening of pig sera for SIV antibodies.  相似文献   

14.
3种禽支原体多重PCR检测方法的建立及应用   总被引：2，自引：1，他引：1  
从鸡毒支原体、鸡滑液囊支原体和火鸡支原体 3种致病性支原体液体培养物中提取DNA,用特定引物分别和组合进行PCR,均得到了特异性扩增产物,片段大小分别为732、207和850 bp。直接用液体培养物进行PCR亦得到了一致的电泳条带。试验结果表明,建立的PCR方法可用于上述3种支原体临床感染的鉴别诊断。ELISA血清学检测和气管拭子PCR检测的比较结果表明,该PCR方法可用于临床MG检测。  相似文献   

15.
In this study, the technical performance of culture, two commercially available polymerase chain reaction (PCR) tests, rapid plate agglutination (RPA) test, hemagglutination inhibition (HI) test, and eight commercially available enzyme-linked immunosorbent assays (ELISAs) were compared for the detection of avian mycoplasma infections from 3 days postinfection (d.p.i.) through 35 d.p.i. The tests were carried out on samples from specified pathogen-free layers that were infected at 66 wk of age with recent Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG) field strains, MS and MG ATCC strains, and Mycoplasma imitans (MIM), respectively. Results showed a high percentage of positive samples in the homologous infected groups and a high percentage of negative samples (100%) in the uninfected and heterologous infected groups during 35 d.p.i. of both culture and PCR tests. For the group infected with the MG 15302 ATCC strain, serology was more sensitive than bacteriology. All MG and MS tests, with the exception of MG ELISA kit D showed a lower percentage of positive samples during 35 d.p.i. for the detection of the MG and MS ATCC strain infection compared with that of the field strains. Also, the number of cross-reactions (false positives) in the serologic tests was lower after infection with an ATCC strain than after an infection with the MG or MS field strain. Contradictory to other studies, the ELISAs and the RPA test using undiluted serum showed a relatively high number of false-positive results. The MG ELISAs (except ELISA kit D) showed more false-positive results (up to 37%) in the MIM-infected group than in the MS-infected groups. This was not unexpected, as MIM and MG have a close antigenic relationship. The results of the serologic tests in this study showed that a certain level of false-positive results can be expected in about any serologic test. Although the level of false-positive results varied between several serologic tests, this study showed that it is not advisable to rely completely on one test (system) only.  相似文献   

16.
A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was established for the detection of antibodies to bovine viral diarrhea virus (BVDV) in bovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified BVDV was used as test antigen at an optimal amount of 1 microgram/well, whereas the optimal concentration of conjugate was at 1/2000 dilution. The standardized test encountered no non-specific reaction with test sera at a starting dilution of 1/10. A total of 50 bovine serum samples was assayed for the presence of antibodies against BVDV by ELISA and serum neutralization test (SNT). A positive correlation between the 2 tests was found. However, ELISA could be as much as 500-fold more sensitive than SNT in detecting low levels of BVDV antibodies.  相似文献   

17.
Yu Z  Jin M  Xu X  Zhang R  Zhou H  Hu Q  Wu H  Qin B  Tan Y  Chen H 《Avian diseases》2006,50(2):264-268
A latex agglutination test (LAT) was developed for rapid detection of antibodies to H5 avian influenza viruses (AIVs). The hemagglutinin protein of H5 AIV was covalently linked to carboxylated latex by ethyl-dimethyl-amino-propyl carbodiimide to prepare the sensitized latex beads. The LAT was evaluated with the hemagglutination inhibition (HI) assay as the reference test. The H5-LAT showed a sensitivity of 87.0% and specificity of 88.9% in detecting 126 serum samples from experimentally infected chickens and a sensitivity of 82.5% and specificity of 86% in detecting 587 field chicken serum samples from mostly vaccinated chickens. The agreement ratio between H5-LAT and HI was found to be 87.3% and 83.1% for the two groups of samples, respectively. Difficulty with background agglutination in stored chicken sera was overcome by serum pretreatment with either dried chicken liver powder or dilution buffer containing detergent Tween-20. The H5-LAT has advantages over a previously reported whole-virus LAT in terms of biosafety in preparation, chemical stability, and higher specificity. It is a rapid and simple test suitable for field monitoring of antibodies to H5-type AIV.  相似文献   

18.
An enzyme-linked immunosorbent assay (ELISA) system was developed for the detection of canine parvovirus (CPV) or CPV antigen in dog faeces and two other ELISA systems were developed for the detection of CPV-specific antibodies in dog sera. The ELISA's were based on the use of CPV-specific mouse monoclonal antibodies, which recognise different epitopes of the haemagglutinin of CPV and which also neutralise the virus. A double antibody sandwich (DAS) ELISA for the detection of CPV in dog faeces was compared with the haemagglutination (HA) test. The DAS-ELISA proved to be more specific, sensitive and easier to perform than the HA assay. An indirect ELISA and a competitive ELISA for the detection of CPV-specific antibodies in dog sera were compared with the haemagglutination inhibition (HI) test. Both ELISA systems proved to be specific and easy-to-use methods for the detection of CPV-specific antibodies. The indirect ELISA, specially, proved to be more sensitive than the HI test. The higher sensitivity and specificity of the ELISA's as compared to HA and HI tests, and their ease of use, make them suitable for routine use in the serology and diagnosis of CPV infections.  相似文献   

19.
A commercial indirect swine influenza virus (SIV) H1N1 enzyme-linked immunosorbent assay (ELISA) was compared with the hemagglutination inhibition (HI) assay by testing 72 samples from experimentally infected pigs and 780 field samples of undefined SIV status. The HI assay was performed using SIV isolates A/Swine/IA/73 for H1N1 and A/Swine/IA/8548-1/98 for H3N2. The ELISA used an SIV isolated in 1988. The results showed that HI and ELISA detected an antibody in 11 and 6, respectively, of 72 serum samples collected from pigs experimentally infected with a 1992 SIV isolate (A/Swine/IA/40776/92). The presence of antibodies in these experimental samples was confirmed by HI tests in which all 72 samples were positive against the homologous virus, a more recent H1N1 SIV isolate (A/Swine/NVSL/01) supplied by National Veterinary Services Laboratories, Ames, Iowa, and a 1999 H1N1 isolate currently used in a commercial vaccine. On testing 780 field samples, an overall agreement of 85.5% was generated between the HI and ELISA. This study demonstrated that the ELISA is a useful serodiagnostic screening test at herd level for detecting swine antibodies against SIV. However, a new SIV isolate representing current SIV strains circulating in the field is needed to replace the older isolates used in the HI and ELISA to increase the test accuracy for serodiagnosis of SIV.  相似文献   

20.
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   

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