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1.
Frey's medium supplemented with artificial liposomes substituting for serum was evaluated for Mycoplasma gallisepticum (MG) serum plate agglutination (SPA) antigen. Antigens prepared in batch (static) culture were compared with antigens grown in a fermenter. All batch-grown MG liposome antigens were highly sensitive, specific, and resulted in a greater yield compared with fermenter-grown liposome antigens. Compared with antigens prepared in Frey's medium with 12% swine serum (regular FMS) or with commercial SPA antigens, liposome antigens had a higher degree of specificity; however, they were similar in sensitivity and antigen yield. The only growth parameter to affect the yield per liter of batch-grown liposome antigen was the concentration of liposomes in the growth medium. The reduced yield and sensitivity of antigens grown in a fermenter may have been due to autoclaving the medium instead of sterilizing by filtration. There was no obvious difference between patterns of serum-medium-grown, liposome-medium-grown, or commercial SPA antigens upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis.  相似文献   

2.
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   

3.
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   

4.
Nonspecific serum plate agglutination reactions to some avian mycoplasma antigens were induced by injecting chickens with several commercial poultry disease vaccines. All of the vaccines were inactivated, and most of them had oil-emulsion adjuvants. The serum plate agglutination reactions appeared within 2 to 3 weeks post-vaccination and generally persisted for several weeks. The plate test reactions were noted with both Mycoplasma gallisepticum (MG) and M. synoviae (MS) antigens, although the degree and duration of the reactions varied with the vaccine involved and the source of MG and MS plate test antigens. Attempts to prevent the nonspecific reactions by heat-inactivation at 56 C for 30 minutes or by addition of equal volumes of solutions of 2-mercaptoethanol, dithiothreitol, or 3 M sodium chloride were ineffective. No hemagglutination-inhibition activity against MG or MS antigens was induced by the vaccines.  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.
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   

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.
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   

9.
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   

10.
Brucella abortus (ADRI 413) was grown on various synthetic, commercial, laboratory-formulated and soil infusion-supplemented agar media. Of 22 media tested, three soil infusion-supplemented media and a serum dextrose agar yielded more brucella antigen than the meat infusion agar, the standard reference medium. Results from agglutination and stability tests indicated that antigens produced from most of the laboratory-formulated media and the soil infusion-supplemented agars compared favourably with that derived from the meat infusion agar. The presence of meat infusion or commercial beef extract appeared to be a basic requirement for producing satisfactory and stable antigen. The growth enhancing effect of certain soil infusions seemed to be associated with the soil organic matter fraction. A significant correlation (p>.05) was obtained between the yield of brucella antigen and the glutamic acid content of the soil infusions. With proper screening, media containing, among other components, beef extract and soil infusions may be of value particularly in areas where the limited supply of fresh meat would make meat infusion agar uneconomical in the production of B. abortus (ADRI 413) antigen.  相似文献   

11.
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   

12.
The sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) for bovine IgG serum antibody to Pasteurella haemolytica was compared with that of an indirect hemagglutination (IHA) test. Pasteurella haemolytica serotypes were grown in a chemically defined cell culture medium, and soluble antigens released into the growth medium were used in the ELISA and IHA test. An ELISA with serotype-1 antigen consistently detected antibody in sera that were positive by IHA test (correlation, 99%). Sera reacting with serotype-1 ELISA antigens also reacted with ELISA antigens prepared from other serotypes. Although ELISA titers averaged 5 log2 units higher than IHA titers, plots of titers determined by the 2 methods were approximately linear. Titer increases detected in paired serum samples by either test were similar. The ELISA was more sensitive than was the IHA in detecting colostral IgG antibody in serum of newborn calves. The ELISA uses a simple, stable antigen preparation and detects antibody to P haemolytica serotypes that commonly infect cattle.  相似文献   

13.
Antibody to smooth Brucella abortus lipopolysaccharide antigen on the surface of polystyrene tubes was detected with peroxidase-labeled antibody against bovine immunoglobulin G. The enzyme-labeled antiglobulin test (ELAT) activity of samples was expressed in arbitrary units/0.01 ml by reference to a standard curve based on tests of dilutions of a positive serum pool. Reactions greater than 3.0 U/0.01 ml were classified positive because specificity at this level was 99.8% (417/418 samples correctly classified negative) with agglutination test-negative sera from 33 Brucella-free herds. Results of the ELAT were compared with results of agglutination tests and the complement-fixation test (CFT), using 430 sera from cattle in 7 infected herds. Activity of greater than 5.0 ELAT U/0.01 ml was detected in all 54 sera classified as positive (titer greater than 1:10) by the CFT, including 5 sera classified as negative by the tube agglutination test. Sera from 8 nonvaccinated cows in the infected herds reacted only by the ELAT, whereas reactions were obtained with 25 and 5 sera by only agglutination tests and the CFT, respectively. The ELAT and CFT results were in agreement for 25 of 26 sera from agglutination test-reactor cattle in herds of unknown status. Comparisons of milk ring and whey agglutination tests with the whey ELAT on 146 quarter samples from cows in an infected herd revealed no ELAT activity greater than or equal to 1.0 U/0.01 ml in the 73 samples considered negative by the 2 other tests. Samples (n = 47) that contained greater than or equal to 1.0 ELAT U/0.01 ml included all (n = 40) samples with milk ring or whey agglutination titers greater than or equal to 1:16 and greater than or equal to 32, respectively, and 7 samples that gave weaker reactions to the latter tests.  相似文献   

14.
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   

15.
The serological responses of dogs to cell wall and internal antigens of B. canis were studied in experimentally infected specific-pathogen-free (SPF) Beagles. Sera from infected and false positive field dogs also were examined. Cell wall antigens were extracted from B. canis by two procedures that employed either hot phosphate buffered saline (PBS) or sodium desoxycholate (SDC). Agar gel immunodiffusion (AGID) tests employing sera from experimentally infected SPF dogs were used to evaluate antigenic extracts. Extraction with PBS yielded two antigens; SDC extracted an antigen complex and sonication of PBS extracted cells liberated four internal antigens.Sera from field dogs that were negative for B. canis infection in repeated tests often had heterospecific antibodies. Such cross-reactive sere commonly gave “spur” (partial fusion) reactions with a positive reference serum when tested against the SDC cell wall antigen. In addition, false positive dogs did not have antibody to one of the cell wall antigens or to the internal antigens. In contrast, sera from infected field dogs commonly gave “identity” (fusion) reactions in the AGID test with two antigens in the SDC extract, and produced precipitin lines to one to four internal antigens.Examination of a library of sera obtained from experimentally infected SPF dogs over a period spanning 412 years revealed that none of the serodiagnostic tests employed (tube agglutination, slide agglutination, AGID) was accurate during the inital 12 weeks of infection; hemocultures were the most sensitive during this period. Tube and slide agglutination tests were initially sensitive, but they showed a lack of sensitivity and specificity after the bacteremic period ceased, as well as in their failure to exclude false positive reactions in field animals. Immunodiffusion tests that employed SDC or PBS extracts of B. canis cell walls were sensitive and accurate in identifying most infected dogs. After the bacteremia had ceased, however, AGID tests that employed cell wall antigens gave equivocal results. Immunodiffusion tests that employed sonicated (internal) antigens were sensitive shortly after the onset of bacteremia, and they had the advantage of detecting infected animals for at least 6 months following the cessation of bacteremia, a time when other serological tests gave equivocal results.  相似文献   

16.
Cell-culture-grown Chlamydia psittaci of turkey origin was treated with phenol and partially purified by differential centrifugation. The most stable antigen/latex mixture occurred with crude antigen precipitated by dimethyl sulfoxide and digested with trypsin. Agglutination reactions occurred within 2 minutes when antigen/latex and antibody-positive serum mixtures were rotated to facilitate contact of the reagents. Nonspecific agglutination of control latex occurred with 1.2% of clinical sera. When C. psittaci was isolated from feces from individual birds, latex agglutination (LA) and direct complement fixation (DCF) together detected antibody activity (titers greater than or equal to 8) in significantly more cases than did DCF alone. It follows, then, that an LA titer alone is highly indicative of a currently active infection or one that has occurred only recently. The isolation rate from birds that had no antibody activity detectable by LA or DCF was 3.8%. In tests on paired clinical sera, LA and DCF agreed in 72.5% of the cases regarding increased, decreased, or stable titers. For detection of antibody activity in single sera, LA had a sensitivity of 39.1% and a specificity of 98.8% relative to DCF detection of antibody activity. The high specificity corroborates the usefulness of LA as an indicator of current or very recent infection.  相似文献   

17.
The dsRNA concentrated polyacrylamide gel electrophoresis (CPAGE) detected rotavirus directly from 19% of 77 stool specimens from diarrheic calves. A commercial enzyme-linked immunosorbent assay (ELISA) detected 25%, latex agglutination test, 23%, and polyacrylamide gel electrophoresis (PAGE), 19%. Establishing CPAGE as the "standard," the commercial ELISA and the latex agglutination test both had higher sensitivity (84%) than PAGE (79%). However, PAGE produced the highest specificity (100%), followed by agglutination (88%) and ELISA (84%). The commercial ELISA had a slightly higher sensitivity than agglutination, PAGE, and CPAGE, but the ELISA specificity was generally lower. The latex agglutination test had a lower sensitivity than ELISA, but specificity was higher. Agglutination had similar negative predictive values (94%), compared with agglutination and PAGe, but had the lowest positive predictive value (a measure of accuracy) (70%). Agreement with CPAGE was highest for PAGE (94.8%), followed by agglutination (87%) and ELISA (84.4%). The calculated percentages of total disagreement with all other tests indicated that ELISA differed from the other rotavirus detection assays in 10.4% of the cases, agglutination in 7.8%, PAGE in 2.6%, and CPAGE in 1.3%. The 2 PAGE assays allowed the detection of atypical rotaviruses from feces based on the characteristic "super-short" migration pattern of the 11 genomic segments of rotaviruses and of other members of the Reoviridae.  相似文献   

18.
Serum antibody responses in feral or domesticated dogs naturally infected with Echinococcus granulosus or/and other common helminths were examined in an enzyme-linked immunosorbent assay (ELISA) using antigens prepared from E. granulosus protoscoleces or oncospheres. The ELISA using the protoscolex antigen was optimised with serums from experimental dogs monospecifically infected with E. granulosus or other helminth parasites, and helminth-free dogs. Anti-protoscolex antibody was detected in 16 of 22 (72.7%) serums from feral dogs with E. granulosus burdens ranging from 300 to 302,600 worms per dog. Seven serums from feral dogs which did not harbour E. granulosus at autopsy but which originated from an endemic hydatid region were tested using protoscolex antigen, and 1 serum gave a positive reaction. One hundred and two serums from dogs known never to have been infected with E. granulosus all gave negative reactions to protoscolex antigen. The sensitivity of the ELISA test proved to be superior to that which has been achieved by arecoline purging as a method of diagnosis for E. granulosus infection in dogs. For use of the assay in hydatid control or eradication campaigns, its sensitivity can be increased by choosing a lower absorbance discrimination value above which serums are regarded as having positive reactions. However, this does introduce positive reactions of some serums from dogs infected with helminths other than E. granulosus. In further development of the assay, use of defined recombinant antigens may improve both sensitivity and specificity.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

19.
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   

20.
Six hundred and thirty-two equine serums were examined for the presence of leptospiral antibodies. A positive reaction to one or more antigenic pools of a formolised leptospiral antigen (used in the rapid macroscopic slide agglutination test) was recorded in 41% of cases.
One hundred samples were tested with 5 formolised antigen pools and 19 live antigens (by the microscopic agglutination test). Of 20 samples in which the live antigen test suggested leptospiral infection with serotypes known to occur in the region, 17 (85%) were confirmed with the formolised antigens.
When the results of both tests were compared, there was agreement in 42 samples (30 positive and 12 negative). Forty-one samples produced equivocal results and 17 gave doubtful reactions to the formolised antigens, 15 of which were negative to the live antigens.
Dilution of the serums 1:1 with normal saline or heat inactivation had no effect in increasing the specificity between the formolised and live antigens. Agreement between operators in the use of the formolised antigen was poor. It is concluded that the formolised antigen has too wide a divergence to be of use for screening horse serums.  相似文献   

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