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1.
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   

2.
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   

3.
Groups of eight chickens were challenged with 10-fold dilutions of one of two strains of Mycoplasma synoviae (MS); each challenge group contained two noninfected sentinels. Both strains were highly efficient in colonizing the respiratory tract with challenge doses as low as 76 and 24 color-changing units/bird. Infection spread rapidly (within 7 days) to sentinels, while uninfected control chickens separated from infected chickens by two empty pens remained uninfected for the 56-day experimental period. Although sentinels and birds challenged with the lowest doses had weaker or slightly slower antibody responses in some cases as measured by serum plate agglutination, enzyme-linked immunosorbent assay (ELISA), and hemagglutination inhibition (HI), they generally exhibited a typical antibody response. Agglutination reactions tended to be weak, but a high percentage of tests (generally >30% from day 14 postchallenge) were positive. ELISA results were variable, and in some cases reactor rates were low (generally <20%), even though the chickens were colonized in the upper respiratory tract. The HI test was reliable in detecting infected groups; usually >50% were positive from 14 days postchallenge. Mean HI titers were higher when using hemagglutination antigens prepared from the homologous MS strain as compared with antigen prepared from the heterologous strain or with standard antigen prepared from WVU 1853.  相似文献   

4.
A dot-immunobinding assay was enhanced by the incorporation of avidin and biotin reagents into the test system (DAB assay). This assay was used to detect serum antibodies to Mycoplasma gallisepticum (MG) and M. synoviae (MS) from chickens. Serum samples were tested by rapid serum plate (RSP), hemagglutination-inhibition (HI), and DAB assay methods. These results were compared. The DAB assay was at least 20 times more sensitive in detecting antibodies for MS and at least 75 times more sensitive in detecting antibodies for MG than the HI test. The DAB assay was as specific as the HI test. The DAB assay was also more sensitive and specific than the RSP test. Some cross-reactions occurred when low dilutions of high-titer sera were used in the DAB assay. Parameters for determining negative, suspicious, and positive samples were established. The DAB assay for MG and MS may have several applications, including use as a screening test and a confirmatory test.  相似文献   

5.
Antibodies in sera and respiratory secretions from chickens infected with Mycoplasma gallisepticum (MG) were measured by an enzyme-linked immunosorbent assay (ELISA). Chickens intratracheally inoculated with 10(5) cells of MG showed a correlation between severity of tracheal lesions and extent of MG colonization in the tracheas in the first 3 weeks postinoculation. Antibody titers in tracheal washings (TWs) of the infected chickens increased during this phase. Thereafter, isolation of MG from the trachea decreased sharply, and there was a concomitant decrease in tracheal lesion scores. At 5 weeks postinfection, the chickens that recovered from the infection exhibited a consistent presence of antibodies in TWs. Chickens reexposed had a faster rate of MG elimination and substantially less severe inflammatory lesions in the tracheas than chickens observed after the first exposure. These findings suggest a possible role of antibodies of the respiratory secretions in resistance to MG. The ELISA was a sensitive and reliable test to detect a minute amount of antibodies in the secretions.  相似文献   

6.
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   

7.
为建立特异性和敏感性高的检验犬细粒棘球绦虫感染的方法。用细粒棘球绦虫(简称,Eg)成虫抗原分别免疫兔和绵羊,收集高免血清,纯化的高免抗体。依据抗体夹心ELISA工作原理,以兔抗体包被,检测感染Eg、不同犬带科绦虫的实验犬和空白犬粪样,绵羊抗体扑捉抗原,HRP标记兔抗绵羊IgG(1∶8 000)催化显色,用酶标仪测定OD 405nm吸光度,用以确定其特异性和敏感性。试验结果表明,敏感性为82.69%(43/52),特异性为85.88%(140/163);粪抗原在感染细粒棘球绦虫16d后可检出,最低抗原浓度为9.7ng/mL即犬感染5条成虫时可检测出阳性。该检测方法具有较好的特异性和灵敏性,为进一步研制检测细粒棘球绦虫虫体抗原ELISA检测试剂盒奠定了基础。  相似文献   

8.
Sensitivity and specificity of 4 different antigen preparations from Leptospira interrogans serovar hardjo were compared in an enzyme immunoassay for detection of antibodies against serovar hardjo type hardjo-bovis in serum. Two antigens prepared using detergents showed serogroup cross-reactivity. A mechanically extracted membrane and a lipopolysaccharide antigen showed a high degree of leptospiral serogroup specificity. The lipopolysaccharide antigen was the most suitable antigen for detection of anti-hardjo antibodies. Enzyme immunoassay was more sensitive than the microscopic agglutination test for detecting antibodies in serum from experimentally and naturally infected cattle. It was not possible to differentiate vaccinated from infected animals or to detect a secondary immune response in vaccinated animals that were subsequently infected.  相似文献   

9.
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.  相似文献   

10.
Enzyme-linked immunosorbent assays (ELISAs) were developed to quantitate hemorrhagic enteritis virus (HEV) antibodies in turkey sera and HEV antigens in tissue extracts. These assays were more sensitive than the commonly used agar-gel precipitin tests in detecting antigen and antibody. The antibody-ELISA was used to monitor the presence and decline of passive antibodies in turkey poults and the seroconversion of turkeys infected with HEV. The antigen-ELISA was carried out using a monoclonal antibody; this test was used to quantitate HEV antigen in experimentally infected turkeys in a time-sequence experiment. Both ELISAs measured a strong antigenic relationship between an avirulent strain (HEV-A) and a virulent strain (HEV-V).  相似文献   

11.
Mycoplasma gallisepticum is a poultry pathogen that causes respiratory disease and loss of egg production worldwide. A live attenuated vaccine, ts-11, has been used for control of M. gallisepticum in several countries. The rapid serum agglutination test is usually used as an indicator of flock response to vaccination; however, in some flocks, the detected response may be weak or absent. With the use of specific monoclonal antibodies against M. gallisepticum strain S6 pMGA in immunoaffinity purification, the major membrane antigen of ts-11 was purified. An indirect enzyme-linked immunosorbent assay (ELISA) was developed with the purified antigen, and its potential for detection of antibodies induced after ts-11 vaccination was compared with an indirect ELISA with M. gallisepticum strain S6 pMGA. In the presence of high levels of ts-11-induced antibodies, both antigens detected similar numbers of positive sera. However, when lower levels of antibodies were present, ts-11 pMGA showed a higher sensitivity than S6 pMGA. Further examination of ts-11 pMGA with Mycoplasma synoviae-infected chicken sera revealed that ts-11 pMGA is specific for M. gallisepticum antibodies. With a panel of sera from ts-11-vaccinated or non-ts-11-vaccinated field chickens, the ts-11 pMGA ELISA was found to be more sensitive than the commercial rapid serum agglutination test in detecting antibodies to ts-11 vaccine. The results from this study suggest that the major membrane antigen of M. gallisepticum may have slightly different antigenic profiles in different strains, thereby necessitating the use of autologous antigens in serodiagnostic assays to increase sensitivity of the tests for mycoplasma antibodies. Thus, the low level of antibody response after ts-11 vaccination is, at least partially, due to the low ability of the current diagnostic antigens to bind ts-11 antibodies.  相似文献   

12.
An enzyme-linked immunosorbent assay (ELISA) was developed in a homologous system with bacterial ultrasonic-treated proteins as the antigen and antisera from chickens infected orally and subcutaneously with the strain Campylobacter jejuni serovar 6 (CJ 6). The cut-off level was determined using antisera from non-infected specific-pathogen-free chickens up to the age of 10 weeks. The suitability of the ELISA system was verified using antisera taken from chickens orally infected at the age of 4 weeks with CJ 1, 6, 28 or 36 or with Campylobacter coli serovar 28 (CC 28). The development of antibodies was monitored up to 6 weeks post-infection (p.i.). Sera from chickens infected with CJ 1, 6, 36 or CC 28 contained specific antibodies to Campylobacter, whereas in those infected with CJ 28 no specific antibodies were found. Distinct cross-reactions were observed between CJ 6, 28 and CC 28 antigens and their antisera 6 weeks p.i., while poor cross-reactions were found with antisera to CJ 1 and 28. Antibodies to strains of all heterologous serovars were successfully detected with an antigen pool comprised of CJ 1, 6 and 36 antigens. In 11 out of the 12 field sera obtained from 5- and 9-week-old broiler chickens suffering from campylobacteriosis, high specific antibody titres to Campylobacter jejuni were found.  相似文献   

13.
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   

14.
Fowl adenoviruses free of avian adenovirus-associated virus, representing 10 serotypes, were tested for cross-reactivity in an enzyme-linked immunosorbent assay (ELISA). All antigens and antisera prepared in chickens, along with uninfected control antigen and normal chicken serum, were reacted in a checkerboard pattern with ELISA. There was considerable cross-reactivity among all serotypes tested. Homologous reactions were generally, but not always, stronger than heterologous reactions. ELISA was about as sensitive as virus neutralization in detecting antibodies. The high sensitivity plus broad-spectrum reactivity should make ELISA a preferred test for the detection of adenovirus antibodies in poultry flocks.  相似文献   

15.
Chen YC  Chen CH  Wang CH 《Avian diseases》2008,52(1):124-129
Many commercial enzyme-linked immunosorbent assays (ELISAs) are unable to differentiate antibody responses to different avian influenza virus (AIV) subtypes. Developing an ELISA for specifically detecting the H5 antibody is the purpose of this study. Four monoclonal antibodies (Mabs) were raised using A/duck/Yunlin/04 (H5N2). They were confirmed as being specific to H5. Two of these antibodies showed hemagglutination inhibition (HI) activity using the HI test. Using immunodot blot assays, three Mabs recognized both Eurasian and American H5, whereas the other Mab recognized only the tested Eurasian H5 virus. When testing denatured H5 antigen, one of the Mabs lost its antigen binding activity using Western blotting. For detecting the H5 humoral response in serum, one monoclonal antibody was purified and labeled with horseradish peroxidase to set up a blocking ELISA. Chicken sera that blocked H5 Mab binding by > 29% were considered H5 antibody positive. Inhibition percentages for sera from chickens infected with other AIV subtypes, H1 to H15, were < 29%. This blocking ELISA was used for 478 field chicken serum samples. The results showed that the sensitivity and specificity of this ELISA were 98.3% (232/236) and 95.9% (232/242), respectively. This blocking ELISA could be used specifically for detecting the H5 humoral responses in chickens.  相似文献   

16.
An enzyme-linked immunosorbent assay (ELISA) was developed in a homologous system with bacterial ultrasonic-treated proteins as the antigen and antisera from chickens infected orally and subcutaneously with the strain Campylobacter jejuni serovar 6 (CJ 6). The cut-off level was determined using antisera from non-infected specific-pathogen-free chickens up to the age of 10 weeks. The suitability of the ELISA system was verified using antisera taken from chickens orally infected at the age of 4 weeks with CJ 1, 6, 28 or 36 or with Campylobacter coli serovar 28 (CC 28). The development of antibodies was monitored up to 6 weeks post-infection (p.i.). Sera from chickens infected with CJ 1, 6, 36 or CC 28 contained specific antibodies to Campylobacter, whereas in those infected with CJ 28 no specific antibodies were found. Distinct cross-reactions were observed between CJ 6, 28 and CC 28 antigens and their antisera 6 weeks p.i., while poor cross-reactions were found with antisera to CJ 1 and 28. Antibodies to strains of all heterologous serovars were successfully detected with an antigen pool comprised of CJ 1, 6 and 36 antigens. In 11 out of the 12 field sera obtained from 5- and 9-week-old broiler chickens suffering from campylobacteriosis, high specific antibody titres to Campylobacter jejuni were found.  相似文献   

17.
Various antigenic extracts of the CU strain of Pasteurella multocida were prepared to determine their suitability as plate antigens for use in the enzyme-linked immunosorbent assay (ELISA) for the detection of fowl cholera antibodies. Antisera from two separate broiler breeder flocks with known fowl-cholera-vaccination histories were collected just before the birds were challenged with virulent strain X-73 P. multocida. A potassium thiocyanate (KSCN)-extracted antigen, a capsular (CAP) antigen, a lipopolysaccharide-protein antigen, and heat-stable, salt-soluble antigen were all suitable as ELISA plate-coating antigens. Filtered and unfiltered sonicates of the CU strain of P. multocida were also suitable ELISA plate antigens. The results suggested that different plate antigens were detecting different populations of antibodies formed in response to fowl cholera vaccinations. When antibody titers were correlated with survival after challenge, the KSCN and the CAP plate antigens placed more nonsurvivors into low-antibody-titer ranges and more survivors (protected birds) into the high-antibody-titer ranges than the other plate antigens.  相似文献   

18.
A double antibody sandwich ELISA for detection of Trypanosoma evansi variant surface glucoprotein (VSG) antigen was established by using two monoclonal antibodies against Trypanosoma evansi VSG antigen.The result of sensitivity showed when positive serum was diluted to 3200 doubles, its D450 nm value was still greater than the critical value of positive and negative,and specificity test result showed that there was no cross reaction with the antigens of Theileria,Toxoplasma gondii,Chlamydia and Fasciola gigantica.82 clinical sera were detected by this double antibody sandwich ELISA and its positive rate was 9.76%.The experiment results demonstrated that this double antibody sandwich ELISA for detection of Trypanosoma evansi VSG antigen had good detecting sensitivity and specificity,and could be used as an effective method for clinical detecting buffalo Trypanosoma evansi disease.  相似文献   

19.
An enzyme-linked immunosorbent assay (ELISA) using sonicated purified Theileria annulata piroplasms was compared with an indirect immunofluorescent antibody test (IFAT) during a vaccination trial in cattle to test different doses and passage numbers of an attenuated T. annulata-infected lymphoblastoid cell-line, and also with Giemsa-stained blood smears during an epidemiological field study of tropical theileriosis in Morocco. The sensitivities of both the ELISA (0.56) and the IFAT using T. annulata piroplasm antigen (0.56) were lower than the IFAT using schizont antigen (0.94) for detecting serum antibodies from 18 cattle immunised 38 days previously with cell-line. The ELISA was, however, the most sensitive test after 180 days (0.50 compared with 0.06 for the piroplasm IFAT and 0.39 for the schizont IFAT), and each test detected antibodies in all sera after challenge with live T. annulata sporozoites. There were minor differences in the ability of blood smear examinations and the ELISA to detect infected and uninfected cattle in the field study at the start and end of the disease season. Initially, the sensitivity and specificity of blood smears were both 0.96 and for the ELISA were 0.83 and 0.86, whereas at the end of the season sensitivity and specificity of blood smears were 0.96 and 0.86 and for the ELISA were 0.95 and 0.94. The specificity of the ELISA was affected by the presence of calves with colostral antibodies, and if these were disregarded the specificities before and after the season were 0.94 and 1.00.  相似文献   

20.
Three batches of strain A5969 Mycoplasma gallisepticum (MG) serum-plate-agglutination (SPA) antigen grown in regular Frey's medium with 12% swine serum, three batches grown in Frey's medium containing artificial liposomes instead of serum, and one commercial SPA antigen were evaluated for sensitivity and specificity. Sensitivity was measured using chickens exposed to MG by intraocular and intranasal inoculation. Specificity was measured in uninoculated controls and in groups inoculated with the oil-emulsion vaccines Haemophilus paragallinarum, infectious bursal disease inactivated virus vaccine, or Staphylococcus aureus. Sera were tested 1 to 8 weeks postinoculation. All SPA antigens had a perfect sensitivity score, except one liposome-grown antigen batch (LC). The two other liposome-grown antigen batches (LA and LB) maintained significantly higher specificity by yielding significantly (P less than 0.01) fewer false positive (FP) agglutination reactions than did the other antigens. The three antigen batches produced in medium with serum had intermediate levels of FP agglutination reactions. When known MG-negative sera were tested, MG SPA antigens LC and commercial SPA antigen yielded significantly (P less than 0.01) higher numbers of FP agglutination reactions than the other SPA antigens.  相似文献   

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