Mycoplasma gallisepticum hemagglutinin V1hA, pyruvate dehydrogenase PdhA, lactate dehydrogenase, and elongation factor Tu share epitopes with Mycoplasma imitans homologues

Mycoplasma gallisepticum is a major pathogen of poultry. Mycoplasma imitans is genetically and antigenically closely related to M. gallisepticum, but so far, only a few proteins of M. imitans have been identified as sharing epitopes with M. gallisepticum. In this study, we identified three proteins of M. gallisepticum that share with M. imitans epitopes defined by monoclonal antibodies (MAbs). MAb 9D4 reacted with the 67-kD hemagglutinin V1hA (previously termed pMGA) of M. gallisepticum and with its continuously expressed 40-kD protein. This MAb also reacted with a 40-kD protein of M. imitans, but not with its putative V1hA. Two-dimensional (2D) immunoblots of M. gallisepticum strains showed that their 40-kD proteins reacting with MAb 9D4 are expressed as major forms with isoelectric points (pI) around 6, and also as less-abundant forms differing in pI. In M. imitans, major forms of 40-kD proteins recognized by MAb 9D4 had pI around 6, whereas minor forms had pI between 5.5 and 5.8. The N-terminal sequence of the M. gallisepticum 40-kD protein recognized by MAb 9D4 strongly indicates that this protein is pyruvate dehydrogenase E1, subunit alpha (PdhA protein, also termed AcoA). The position of elongation factor Tu (EF-Tu), detected by the reference MAb GB8, was very similar in the 2D proteome maps of M. gallisepticum and M. imitans (MW of about 45 kD; pI - 5.6). In both M. gallisepticum and M. imitans, MAb 7G1 reacted with proteins of about 36 kD with similar charges (major forms with pI of about 8). The position of this protein in the proteome map of M. gallisepticum and its N-terminal sequence strongly suggest that MAb 7G1 recognizes lactate (malate) dehydrogenase (Ldh or Mdh). Comparison of 2D proteomes of 10 M. gallisepticum strains indicated that positions of EF-Tu, PdhA, and Ldh proteins are rather consistent and can be used as reference points in further analyses of the M. gallisepticum proteome.     

Monoclonal antibodies that recognize specific antigens of Mycoplasma gallisepticum and M. synoviae

The polypeptide profiles of the type strains of Mycoplasma gallisepticum (PG 31) and M. synoviae (WVU 1853) resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were compared. Except for a few discrete peptides that were similar, the species varied considerably in peptide profiles. Congruence was observed between the type strains of each species and homologous cloned serotypes. Protein blots of each species were probed with 2 mouse monoclonal antibodies. Monoclonal antibody G 46 was specific for the antigen p 110 (G) in M. gallisepticum, and S 221 was specific for an antigen complex p 45-50 (S) in M. synoviae. The 2 monoclonal antibodies clearly distinguished between all serotypes of M. gallisepticum and M. synoviae that were examined by Western blot transfer. Autoradiographs of 125I-labeled M. gallisepticum and M. synoviae indicated that p 110 (G) and p 45-50 (S) were surface membrane peptides. Indirect immunofluorescence of M. gallisepticum and M. synoviae in Vero cell cultures supported the autoradiographic findings. The p 110 (G) antigen of M. gallisepticum was heat-stable, pronase-sensitive, and resistant to periodate oxidation, suggesting that its chemical composition is protein. In contrast, the p 45-50 antigen complex of M. synoviae appeared as a broad band in protein blots treated with monoclonal antibody S 221, was sensitive to pronase, and responded to Schiff's reagent but was not completely inhibited by periodate oxidation, suggesting that it is a complex of repeating sequences probably composed of glycosylated peptides.     

Phenotypic variation of Mycoplasma iowae surface antigen

The diseases caused by mycoplasmas are generally chronic and persistent and apparently occur notwithstanding a host immune response. The ability to evade the host immune response is facilitated by phenotypic variation in the mycoplasma surface components. The avian mycoplasmas Mycoplasma gallisepticum and Mycoplasma synoviae have both been previously described to be able to switch their surface antigens, and recent evidence indicates that M. gallisepticum could switch surface antigens under natural conditions in vivo. In this work we present data to support that Mycoplasma iowae is also capable of phenotypic variation as detected by reactions with the monoclonal antibody MAb MI1 on colony immunoblots. This phenomenon is suggested to occur spontaneously or in response to normal serum and tissue components and may play an important role in the evasion of the host immune response.     

Cloning and analysis of the gene for a major surface antigen of Mycoplasma gallisepticum

Myplasma gallisepticum infects a wide variety of gallineaceous birds including chickens, turkeys, and pheasants. Infection occurs both horizontally and vertically. Thus, control of the spread of M. gallisepticum to noninfected flocks is difficult. Continual monitoring is necessary to identify infected flocks even under the most stringent infectious control practices. Monitoring, however, is usually performed by measuring hemagglutination activity (HA) in serum, an insensitive and variable test. Variability in the HA test arises differences in agglutination antigen, changes in antigenic profiles of the M. gallisepticum strain, and variability in reading the agglutination reaction. Enzyme-linked immunosorbent assays (ELISAs) are the preferred method of testing because of the ease in obtaining sera and the sensitivity and reproducibility of the assays, but the ELISA suffers from a lack of standardization in the test antigen. The ELISA test will be more easily accepted once the test antigen has been standardized. To this end, we have identified, cloned, and characterized the gene for an antigen that has potential as a species-specific antigen for M. gallisepticum The gene codes for a 75-kD protein, P75, that is recognized during natural infections. Recombinant P75 is not recognized in immunoblots by convalescent sera produced in chickens infected with Mycoplasma synoviae, Mycoplasma gallinarum, and Mycoplasma gallinaceum or in turkeys infected with Mycoplasma meleagridis.     

Haemadsorption inhibition test for the identification of Mycoplasma gallisepticum and Mycoplasma synoviae

Summary Colonies of the avian mycoplasma strains Mycoplasma gallisepticum S6 and Mycoplasma synoviae WVU 1853 and two Mycoplasma synoviae isolates from this laboratory were shown to be haemadsorption positive for chicken erythrocytes. Three Mycoplasma synoviae isolates from this laboratory proved to be haemadsorption and haemagglutination negative. The haemadsorption of the mycoplasma colonies mentioned above was inhibited with specific antisera of either high or low titre. No cross-inhibition was observed. It is suggested that this test could be used for a quick tentative identification of the two avian mycoplasmas on primary solid-medium cultures.     

Haemadsorption inhibition test for the identification of Mycoplasma gallisepticum and Mycoplasma synoviae

Summary

Colonies of the avian mycoplasma strains Mycoplasma gallisepticum S6 and Mycoplasma synoviae WVU 1853 and two Mycoplasma synoviae isolates from this laboratory were shown to be haemadsorption positive for chicken erythrocytes. Three Mycoplasma synoviae isolates from this laboratory proved to be haemadsorption and haemagglutination negative. The haemadsorption of the mycoplasma colonies mentioned above was inhibited with specific antisera of either high or low titre. No cross‐inhibition was observed. It is suggested that this test could be used for a quick tentative identification of the two avian mycoplasmas on primary solid‐medium cultures.     

The humoral immune response of chickens to Mycoplasma gallisepticum and Mycoplasma synoviae studied by immunoblotting

The humoral immune response over time of White Leghorn chickens experimentally infected with Mycoplasma gallisepticum or M. synoviae by an aerosol inoculation or a contact exposure were compared by immunoblotting. The response of chickens infected with M. gallisepticum were similar with respect to proteins recognized and intensity of response, regardless of mode of infection. On the other hand, chickens infected by aerosolization of M. synoviae responded to more proteins and with greater intensity than did M. synoviae contact-exposed birds. Chickens infected with M. gallisepticum responded with antibodies to over 20 proteins, while chickens infected with M. synoviae responded with antibodies to 12 proteins. Field sera from chickens naturally infected on commercial poultry farms with M. gallisepticum or M. synoviae were analyzed by immunoblotting and were found to react with a number of mycoplasma proteins. However, no correlation was seen when comparing intensity of immunoblot staining and hemagglutination-inhibition titer of the field sera. The experimental antisera were used to identify species-specific proteins of M. gallisepticum and M. synoviae. Six immunogenic species-specific proteins of M. gallisepticum with relative molecular masses of 82 (p82), 65-63 (p64), 56 (p56), 35 (p35), 26 (p26), and 24 (p24) kilodaltons (kDa) were identified. Two species-specific proteins of M. synoviae with relative molecular masses of 53 (p53) and 22 (p22) kDa were identified. Additionally, a highly immunogenic 41 (p41) kDa protein of M. synoviae was identified. Species-specific proteins identified in these mycoplasmas and the 41 kDa protein of M. synoviae were purified by preparative SDS-PAGE in amounts sufficient for further characterization and for use in serodiagnostic tests.     

Triton X-100-solubilized Mycoplasma gallisepticum and M. synoviae ELISA antigens

Triton X-100-solubilized Mycoplasma gallisepticum and M. synoviae ELISA antigens were found to be more specific and sensitive than six other antigens at a concentration of 250 ng protein/0.1 ml per microtiter plate well.     

A coagglutination assay with monoclonal antibodies for rapid laboratory identification of Mycoplasma synoviae.

An agglutinating monoclonal antibody (MAb S2) specific for a 55,000-molecular-weight surface protein of Mycoplasma synoviae was developed by fusion of spleen cells from immunized BALB/c mice with P3X63 Ag8.653 myeloma cells. Immunogold labeling experiments confirmed the cell surface location of the MAb-recognized antigen. MAb S2-coated Staphylococcus aureus was used in a rapid slide coagglutination assay. Eleven M. synoviae strains, including the type strain WVU 1853, coagglutinated with MAb-coated S. aureus, but five M. gallisepticum strains (PG31, S6, R, F, and A5969) did not.     

Immunohistochemical detection of Mycoplasma gallisepticum antigens in turkey respiratory tissues

An avidin-biotin-immunoperoxidase diagnostic test was developed to facilitate rapid identification of Mycoplasma gallisepticum in respiratory tissues of turkeys. This procedure used polyclonal primary antibodies produced in rabbits. Turkeys were inoculated into the infraorbital sinus and trachea with the R strain of M. gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis, or Frey's media. The outer walls of the infraorbital sinuses, lungs, and tracheas were collected and fixed in either 10% neutral formalin or pentanedial methyl glycol at 1, 2, 3, and 4 wk postinoculation. Tissues were subdivided and remained in each fixative for 6 or 24 hr. The avidin-biotin-immunoperoxidase diagnostic test was sufficiently sensitive to detect M. gallisepticum antigen at 1, 2, 3, and 4 wk postinoculation. Staining of M. gallisepticum was significantly more intense on infraorbital sinus epithelium than on respiratory epithelium from the trachea or lung. Statistical analysis indicated that the 6-hr fixation time offered better antigen preservation than 24 hr in a fixative. There was no difference in intensity of M. gallisepticum antigen staining in tissues fixed in methyl pentanedial glycol when compared with tissues fixed in 10% neutral buffered formalin. Significant differences in staining intensity were observed between weeks. Specificity of the avidin-biotin-immunoperoxidase test was not complete. None of the tissues from the M. meleagridis and control groups showed staining. No staining was observed in the ciliated brush border of infraorbital sinus epithelial cells from turkeys infected with M. synoviae. However, weak to moderate staining was observed in several tracheas of turkeys inoculated with M. synoviae. Improved specificity of an avidin-biotin-immunoperoxidase diagnostic test to detect M. gallisepticum in respiratory tissues of turkeys probably will require the use of multiple monoclonal antibodies directed against several different epitopes specific to the cell membrane of M. gallisepticum.     

Identification of Mycoplasma gallisepticum by use of monoclonal antibody in a rapid slide agglutination test.

Monoclonal antibody (MAb) against Mycoplasma gallisepticum strain PG31 was produced in BALB/c mice. The MAb (designated M9) was of IgG3 isotype and reacted with an epitope in M gallisepticum antigens with molecular weights of 35, 90, 95, and 98 kilodaltons (kDa). The M9 reacted with M gallisepticum antigens in the dot-blot ELISA and in western blot assays. It agglutinated M gallisepticum strains PG31, F, R, S6, A5969, and 9 field isolates from various sources. A coagglutination assay, using Staphylococcus aureus (Cowan strain 1), was developed to enhance the agglutination of some weakly agglutinating M gallisepticum isolates. The M9 did not react with M synoviae, M iowae, M meleagridis, M gallinarum, or M gallinaceum in any of the aforementioned assays. This MAb may be useful in facilitating laboratory diagnosis of M gallisepticum infections.     

In vitro susceptibilities to fluoroquinolones in current and archived Mycoplasma gallisepticum and Mycoplasma synoviae isolates from meat-type turkeys

Monitoring of susceptibility to antibiotics in field isolates of pathogenic avian mycoplasmas is important for appropriate choice of treatment. Our study compared in vitro susceptibility to enrofloxacin and difloxacin in recent (2005-2006) isolates of Mycoplasma gallisepticum and Mycoplasma synoviae from meat-type turkey flocks with archived (1997-2003) isolates and reference strains. Comparison of minimal inhibitory concentration (MIC) values determined by microtest, agar dilution and commercial Etest showed good agreement, but underscored the need for standardized methods for testing. Notably, while the commercial Etest was convenient and accurate for determining MICs for enrofloxacin in the range 0.002-0.094mug/ml, the endpoint of inhibition for M. gallisepticum and M. synoviae strains with MIC values >/=1.0mug/ml could not be determined. A decrease in susceptibility to both fluoroquinolones was detected in archived strains but to a greater degree in recent isolates, most of which had MICs above the NCCLS susceptibility breakpoint for these antibiotics (相似文献   

Detection of Mycoplasma gallisepticum by direct immunofluorescence using a species-specific monoclonal antibody

A monoclonal antibody against Mycoplasma gallisepticum (MG) (strain S6) was prepared in mice and identified as isotype IgG1 by standard procedures. Although it did react at high titers (1:100,000) in the enzyme-linked immunosorbent assay (the original method for its identification), it failed to react in the agglutination, hemagglutination-inhibition, and growth-inhibition tests. When conjugated to fluorescein isothiocyanate, the monoclonal antibody reacted with the homologous and eight "atypical" strains of MG but not with M. meleagridis or M. synoviae in the direct fluorescent-antibody test. This reagent may be useful for detecting field infections involving atypical strains of MG.     

Identification of major immunogenic proteins of Mycoplasma synoviae isolates

Mycoplasma synoviae isolates differ in patterns of immunogenic proteins, but most of them have not been identified yet. The main aim of this study was their identification in two closely related M. synoviae isolates, ULB 02/P4 and ULB 02/OV6, recovered recently from chickens in Slovenia. N-terminal sequencing identified 17 M. synoviae proteins. Amongst them were 14 major, highly expressed but previously unidentified proteins, including enzymes, chaperones and putative lipoproteins. ULB 02/P4 proteins with increasing molecular weight (M(w)) in the region above the lipoprotein MSPB (approximately 40 kDa) were elongation factor EF-Tu, enolase, NADH oxidase, haemagglutinin MSPA, ATP synthase beta chain, trigger factor, pyruvate kinase and chaperone DnaK. Enolase (approximately 47 kDa) seemed to be immunogenic for chickens infected with M. synoviae, whereas EF-Tu, which might cross-react with antibodies to the P1 adhesin of Mycoplasma pneumoniae, was not. ULB 02/OV6 synthesized several immunogenic proteins and those with M(w) of approximately 70, 78, 82, 90, 110 and 160 kDa, cross-reacted with antibodies to Mycoplasma gallisepticum. They remain to be identified, because besides putative lipoproteins, protein bands of 78, 82, 85 and 110 kDa contained also dehydrogenase PdhD, elongation factor EF-G, enzyme PtsG and putative neuraminidase, respectively.     

鹅败血支原体及滑液囊支原体感染的血清学调查

本研究对珠江三角洲部分地区鹅群的185例血清样品，进行败血支原体、滑液囊支原体感染的平板凝集试验，检测结果表明：鹅败血支原体的感染率为29.7%，滑液囊支原体的感染率为19.5%，两者的混合感染率为14.6%。     

Genetic and antigenic relatedness between Mycoplasma gallisepticum and Mycoplasma synoviae

The two avian pathogens Mycoplasma gallisepticum and Mycoplasma synoviae were found, by Southern blot hybridization of their digested DNAs, to share genomic nucleotide sequences additional to those of the highly conserved ribosomal RNA genes. The assumption that some of the shared sequences encode for antigens or epitopes common to both mycoplasmas was supported by Western immunoblot analysis of cell proteins of one mycoplasma with specific antiserum to the other mycoplasma. Interestingly, the band patterns of reactive antigens were different for some of the M. gallisepticum strains, supporting the concept that the species is genotypically variable. The results of the present study may explain the cross-reactivity of the two mycoplasmas noted previously in a variety of routine serological tests.     

In vitro development of resistance to enrofloxacin,erythromycin, tylosin,tiamulin and oxytetracycline in Mycoplasma gallisepticum,Mycoplasma iowae and Mycoplasma synoviae

The in vitro emergence of resistance to enrofloxacin, erythromycin, tylosin, tiamulin, and oxytetracycline in three avian Mycoplasma species, Mycoplasma gallisepticum, Mycoplasma synoviae and Mycoplasma iowae was studied. Mutants were selected stepwise and their MICs were determined after 10 passages in subinhibitory concentrations of antibiotic. High-level resistance to erythromycin and tylosin developed within 2-6 passages in the three Mycoplasma species. Resistance to enrofloxacin developed more gradually. No resistance to tiamulin or oxytetracycline could be evidenced in M. gallisepticum or M. synoviae after 10 passages whereas, resistant mutants were obtained with M. iowae. Cross-sensitivity tests performed on mutants demonstrated that mycoplasmas made resistant to tylosin were also resistant to erythromycin, whereas mutants made resistant to erythromycin were not always resistant to tylosin. Some M. iowae tiamulin-resistant mutants were also resistant to both macrolide antibiotics. Enrofloxacin and oxytetracycline did not induce any cross-resistance to the other antibiotics tested. These results show that Mycoplasma resistance to macrolides can be quickly selected in vitro, and thus, providing that similar results could be obtained under field conditions, that development of resistance to these antibiotics in vivo might also be a relatively frequent event.     

Identification of the antigenic components of the virulent Mycoplasma gallisepticum (R) in chickens: their role in differentiation from the vaccine strain (F)

The antibody response to different proteins of Mycoplasma gallisepticum (MG) was studied in chickens experimentally infected with virulent MG R strain. The chickens were challenged at 8 weeks of age by the intranasal route. Each cockerel received 1.3 X 10(6) colony-forming units (CFU). MG strains (R and F) were banded by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The banding pattern was distinctively different between the two strains in the range of 92.5 to 200 kilodaltons (kD). Chicken sera collected at different times following challenge were analyzed by Western blot to determine the patterns of antibodies raised to specific MG proteins (R versus F strains). Early in infection (2 weeks postchallenge), antibodies to 60-kD and 75-kD polypeptides of MG R strain were produced. Subsequently (greater than or equal to 4 weeks postchallenge), antibodies recognized a larger number of MG antigens in both strains. The immunoblot patterns remained the same in the period 8-11 weeks postinfection in each of the two strains; however, the patterns were different when the two strains were compared. The early response recognized the 75-kD protein in the R strain while it recognized the 80-kD protein in the F strain. The late response recognized the 130-kD protein and the protein slightly heavier than 200 kD in the R strain. These two bands did not appear in the immunoblot performed against the F strain of MG. Electroeluted protein of MG R strain, namely adhesin (75 kD), showed a hemagglutination activity (HA) on chicken red blood cells. With the appearance of antibodies specific to the 60-kD and 75-kD polypeptides, there was a significant rise in hemagglutination-inhibition geometric mean titer of chicken sera.     

Comparison of Mycoplasma gallisepticum subunit and whole organism vaccines containing different adjuvants by western immunoblotting

Chickens were vaccinated with subunit (adhesin protein) or whole organisms of Mycoplasma gallisepticum (MG) adjuvanted with multilamellar positively charged liposomes or oil-emulsion. Sera were collected before and following the first (13 weeks of age) and second (17 weeks of age) vaccination. The chicken sera were used in western immunoblotting against whole MG polypeptides. Vaccination with the subunit (MG-adhesin) bacterin containing positively charged liposomes resulted in antibody response specific to adhesin band (75 kD) at 3 weeks post the first and second vaccination; however, crossreactions of the same antibodies occurred to MG proteins of 85 kD (3 weeks after the first vaccination) and 56 kD (3 weeks after the second vaccination). Vaccination with whole MG proteins containing positively charged liposomes resulted in significant immunopotentiation of antibodies against low molecular weight polypeptides of MG (less than 48.0 kD). The addition of Salmonella typhimurium cell wall proteins mitogens (STP) to the different bacterins suppressed the antibody responses to some MG polypeptides.     

以重组PvpA蛋白作为抗原的鸡毒支原体抗体间接ELISA检测方法的建立

为建立鸡毒支原体(MG)种特异性检测的间接ELISA检测方法,本研究以原核表达的PvpA蛋白作为包被抗原,初步建立了检测MG抗体的间接ELISA检测方法.特异性试验结果表明,重组PvpA蛋白与鸡新城疫病毒、鸡传染性支气管炎病毒、滑液支原体、大肠杆菌“O”抗原、禽沙门氏茵“O”抗原阳性血清均不发生交叉反应.该方法的批内变异系数小于8％,批间变异系数小于11％,表明具有较好的重复性.采用该检测方法与进口试剂盒对不同省份送检的鸡血清进行检测比较,两者阳性和阴性符合率均达到90％以上.本研究建立的间接ELISA检测方法为MG抗体检测试剂盒的研制奠定了基础.