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1.
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   

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.
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   

4.
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   

5.
Liu M  Guo S  Hu S  Xiao Y  Xu Q  Li Z  Bi D  Sun M 《Veterinary microbiology》2008,130(1-2):99-106
The S-layer protein CTC surface-display system of Bacillus thuringiensis (Bt) was used to test the possibility of displaying the protein of Mycoplasma gallisepticum (MG) agglutinin (pMGA) on the Bt cell surface. By fusing part of pmga1.2 (pmga1.2p) with the surface-anchoring motif of ctc, two recombinant plasmids, pCTC-PMGA1.2P and pCSPMGA1.2P, were constructed. They harboured the fusion genes ctc-pmga1.2p and csa-ctc-pmga1.2p (csa represents csaAB operon, important in anchoring the S-layer protein on the cell surface), respectively. Two recombinant Bt strains were constructed by electro-transferring recombinant plasmids to a Bt plasmid-free derivative strain BMB171. Strains obtained were BCCG (bearing pCTC-PMGA1.2P and the csaAB operon-carrying plasmid pMIL-CSA) and CG (pCSPMGA1.2P). The vegetative cells of both strains were used as antigens for haemagglutination (HA) and haemagglutination inhibition (HI) assays. HA and HI assays showed that recombinant PMGA1.2P proteins were not only displayed on the cell surface of BCCG and CG, but also specific to MG-positive serum. After oral immunization of chickens with spores, both BCCG and CG elicited a humoral response to PMGA1.2P and exhibited immunogenicity, as indicated by serum plate agglutination (SPA) assays. This study suggests the possibility of generating heat-stable and oral vaccines against infectious diseases of fowl with Bt surface-display system.  相似文献   

6.
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   

7.
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.  相似文献   

8.
Sera from chickens inoculated with various challenge infectious bursal disease viruses or infectious bursal disease vaccines were found to cross-react in the Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) serum plate agglutination (SPA) tests. Two-fold dilutions of these cross-reacting sera with phosphate-buffered saline before retesting eliminated all non-specific agglutination in the MG and MS SPA tests. Cross-reactions were observed in the SPA test using sera from chickens inoculated with either MG or MS. Dilutions of these sera 1:2 had little effect on the number of these cross-reactions. At 1:4 serum dilutions, however, the number of cross-reactions between MG and MS was reduced. At 1:8 dilution of test sera, cross-reactions between MG and MS were further reduced. Some reduction in specific MG and MS SPA reactions, however, also occurred at the 1:8 dilution of sera with some of the plate antigen.  相似文献   

9.
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   

10.
Six groups of ten 18-week-old mycoplasma-free white leghorn pullets were vaccinated with one of the following: Mycoplasma gallisepticum (MG) bacterin. Haemophilus gallinarum bacterin, Pasteurella multocida bacterin, combined infectious bursal disease (IBD)-Newcastle (NDV) chicken-embryo-origin (CEO) vaccine. IBD-NDV tissue-culture-origin (TC) vaccine, or saline emulsified in oil; one group received no vaccine. Plate agglutination tests for M. synoviae (MS) and MG were done for 10 weeks after vaccination using three different test antigens. Pullets vaccinated with H. gallinarum bacterin and IBD-NDV TC vaccine showed the greatest incidence of nonspecific plate agglutination reactions. The incidence of positive plate agglutination reactions varied with test antigens. Five groups of fifty 18-week-old mycoplasma-free heavy-breed pullets were vaccinated with one of the following: saline emulsified in oil, chicken embryo fibroblasts emulsified in oil, allantoic fluid emulsified in oil, chicken embryos emulsified in oil, or MS-contaminated chicken embryos emulsified in oil. Plate agglutination tests for MS and MG were done for 8 weeks after vaccination. Chickens vaccinated with chicken embryo fibroblasts emulsified in oil had the greatest incidence of nonspecific plate agglutination reactions. Pullets vaccinated with MS-contaminated chicken embryo vaccine had only a small increase in MS-positive plate agglutination reactions compared with pullets vaccinated with uncontaminated chicken embryo vaccine.  相似文献   

11.
Specific pathogen free hens and males were experimentally infected with Mycoplasma gallisepticum. Eggs were then collected, and a part was incubated and set for hatching. Mycoplasma cultures were performed on infected adults and antibodies to MG were analysed by use of slide agglutination (SA) test and commercial ELISA tests on adults and chicks sera and on yolks from non incubated eggs. Both ELISA tests could detect antibodies in yolks from non incubated eggs laid three weeks after infection. SA and the three ELISA tests revealed positive sera in chicks hatched from eggs laid as soon as one week after infection.  相似文献   

12.
Serum and yolks from commercial flocks and from hens exposed to Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and Mycoplasma gallisepticum (MG) were tested for immunoglobulin G antibody by the enzyme-linked immunosorbent assay (ELISA) and the hemagglutination-inhibition (HI) test. Yolks prepared by chloroform extraction and low-speed centrifugation performed well in the serological tests used and were a suitable alternative to serum for antibody determination by the ELISA for NDV, IBV, and MG and by HI test for NDV.  相似文献   

13.
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   

14.
Three-week-old turkeys were inoculated intranasally with approximately 10(6) colony-forming units (CFU) of putative variant Mycoplasma gallisepticum (MG) strains M876, M35, or the virulent S6 reference strain. Uninoculated turkeys in each group served as contact sentinels. The hemagglutination-inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) were used to determine serologic responses. MG was isolated from 100% and 92% of S6- and M876-inoculated turkeys, respectively, on day 7 PI. However, culture-positive rates among M876-inoculated turkeys declined more rapidly, transmission to contact sentinels took longer and occurred at lower rates, and serologic responses measured by HI and ELISA were lower than in S6-infected turkeys. Testing sera from inoculated turkeys for antibodies to MG in homologous and heterologous ELISA systems indicated that strain M876 was significantly (P less than 0.05) less immunogenic than S6 (days 62 and 95 PI), and that the homologous ELISA was more sensitive (P less than 0.005). MG strain M35 failed to infect turkeys in three attempts, even though the inocula used were viable on culture media.  相似文献   

15.
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16.
Broiler minibreeder hens were used to produce monovalent antisera to bacterins prepared from serotypes 1, 3, 4, and 3 X 4 cross (CU strain) of P. multocida and to a polyvalent fowl cholera bacterin containing serotypes 1, 3, and 4. Antiserum to the CU strain (live vaccine) was also produced. Monovalent enzyme-linked immunosorbent assay (ELISA) plate antigens were prepared by separately sonicating each of the strains. Polyvalent plate antigen (Poly 3) was prepared by combining, in equal amounts after sonication, antigens from serotypes 1, 3, and 4. Each antiserum was assayed against its homologous ELISA plate antigen and against all other heterologous plate antigens, including Poly 3. The strongest reactions, as indicated by the highest absorbance values, were observed in homologous ELISAs. The CU strain may be the best monovalent ELISA plate antigen for detecting antibodies formed in response to a commercial polyvalent bacterin and to vaccinations with the live CU strain. Overall, monovalent serotype 1 (strain X-73) antiserum did not react well with any other heterologous ELISA plate antigen, whereas monovalent antisera of serotypes 4 (strain P-1662) and 3 X 4 (CU strain) reacted equally strongly with monovalent serotype 4 ELISA plate antigen. Background binding of negative serum was significantly lower (P less than 0.05) when using CU plate antigen than when using any of the other plate antigens.  相似文献   

17.
Since 1994, an epidemic of conjunctivitis caused by Mycoplasma gallisepticum (MG) has spread throughout the eastern population of house finches (Carpodacus mexicanus). The adaptation of MG to a free-flying avian species presents potential problems for the control of mycoplasmosis in commercial poultry. To evaluate risks associated with this emerging problem, a field survey was conducted to assess prevalence of MG infection in house finches and other passerine birds associated with poultry farms. Between November 1997 and March 1999, 1058 birds were captured by mist net or trap at 17 farms and at 10 feeder stations in northeast Georgia. Birds were bled and screened by serum plate agglutination (SPA) for antibodies to MG. Birds with negative or weak positive SPA results were released at capture sites, and those with strong positive SPA reactions were kept for further evaluation. Necropsies were performed on selected house finches and individuals of 11 other passerine species, and samples were collected for MG testing by culture, polymerase chain reaction (PCR), hemagglutination inhibition, and histopathology. Testing revealed 19.1% of 671 birds caught at farms and 11.6% of 387 birds caught at feeder sites were SPA positive for MG. Three house finches captured on farms were positive for MG by culture and PCR, whereas three from feeder sites were positive only by PCR. No MG isolates were made from tufted titmice (Baeolophus bicolor), but 40% were positive by PCR. Individuals from 10 additional species were SPA positive only. Results suggest that MG persists at low levels in house finches in northeast Georgia and that tufted titmice may be nonclinical carriers of MG or a related mycoplasma. Positive SPA reactions in other species may be caused by nonspecific reactions or contact exposure. Current biosecurity recommendations should be sufficient to minimize risks of transmission between wild and domestic birds.  相似文献   

18.
Serologic testing by the serum plate agglutination (SPA) procedure was performed to detect the presence of cross-reacting antibodies to Mycoplasma meleagridis, Mycoplasma synoviae, and Mycoplasma gallisepticum in lesser prairie-chickens (Tympanuchus pallidicinctus) trapped over a 2-yr period in Finney and Kearny counties of southwestern Kansas. Sera examined from birds (n = 50) obtained in March-April 2000 tested positive for M meleagridis, M. synoviae, and M. gallisepticum at levels of 6%, 10%, and 10%, respectively, for the population examined. Mycoplasma meleagridis antibodies were detected in 3 samples (2.7%), M. synoviae antibodies in 2 samples (1.7%), and M. gallisepticum antibodies in 3 samples (2.7%) from birds (n = 112) collected in March-April 2001. Data obtained by SPA can result in false positives and should be verified by additional procedures such as the hemagglutination-inhibition test. Low amounts of sera prohibited this additional testing. Thus, the positive SPA results should be considered presumptive for the presence of Mycoplasma antibodies. Although Mycoplasma antibodies have been detected in wild turkeys (Meleagris gallopavo) from Kingman and Butler counties in Kansas, this report is the first of possible mycoplasmosis in Finney and Kearny counties, Kansas. All birds testing positive by this procedure should be considered as potential carriers of Mycoplasma and should not be used in relocation efforts.  相似文献   

19.
A simple adhesion-hemadsorption inhibition (AHAI) test was developed for the detection of antibodies to Mycoplasma gallisepticum in the chicken sera. The AHAI antibody was detected simultaneously with HI antibody from sera of chickens intratracheally inoculated with viable cells of M. gallisepticum. A good correlation between HI and AHAI antibody titers was obtained with 382 (84.7%) of 451 sera from chickens reared on farms spontaneously contaminated with M. gallisepticum, whereas the remainder, 69 sera, was positive for HI but negative for AHAI test. It was not apparent whether the latters exhibited a non-specific reaction or the discrepancy was due to the lower sensitivity of AHAI reaction. The AHAI test does not require a great amount of antigen, special reagents or instruments, or pre-absorption treatment of test sera, and, therefore, it may serve as a simple serological test for detecting antibodies to M. gallisepticum.  相似文献   

20.
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   

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