A survey of avian Mycoplasma species for neuraminidase enzymatic activity

Among 23 currently recognized avian Mycoplasma (AM) species only Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis and Mycoplasma iowae cause disease and loss of production in chickens and/or turkeys. Because neuraminidases are considered virulence factors in many pathogenic microorganisms the aim of our study was to determine which AM species possess neuraminidase enzymatic activity (NEAC). Small samples of AM cells were assayed for NEAC using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-alpha-d-N-acetylneuraminic acid. In the case of positive NEAC reaction the substrate gave the insoluble indigoblue product what enabled simple test and easy estimation of NEAC. M. gallisepticum and M. synoviae which share sequences of the gene encoding neuraminidase (sialidase NanH) exhibited considerable levels of NEAC. However, NEAC levels differed among their strains, as well as among cultures of different strains. Only certain cultures of the type strain of M. meleagridis showed NEAC, whereas among six serovars of M. iowae only serovar I (type strain 695) showed NEAC. Weak NEAC was detectable in M. anseris, M. cloacale and M. pullorum, whereas the type strain of M. corogypsi (BV1) showed strong NEAC. Our study provides novel informations about NEAC in AM species and suggests that higher invasiveness and possibly, the pathological processes might be associated with their NEAC.     

Sialidase activity in Mycoplasma synoviae

Eleven strains of the avian pathogen Mycoplasma synoviae were evaluated for the presence of sialidase activity with the use of the fluorogenic substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and the sialidase inhibitor 2-deoxy-2,3- didehydro-N-acetylneuraminic acid. The kinetics of in vitro growth in modified Frey medium were also assessed for each strain. Five strains had been isolated from clinically symptomatic chickens, and strains WVU 1853T and K3344 have been demonstrated to be capable of reproducing disease in specific-pathogen-free chickens. All strains exhibited sialidase activity, although the amount varied 65-fold among strains (P < 0.0001) from 1.3 x 10(-7) to 2.0 x 10(-9) activity units per colony-forming unit. Strains originally isolated from clinically symptomatic birds had more (P < 0.05) sialidase activity than strains from asymptomatic birds. Strain WVU1853T exhibited the most sialidase activity (P < 0.0001) and grew to the highest culture density (P < 0.0001) among strains, but across strains, the rank correlation of growth rate with sialidase activity was not significant. Negligible activity was detected in conditioned culture supernatant fluid. This is the first report of sialidase activity in pathogenic strains of M. synoviae, which suggests a potential enzymatic basis for virulence of the organism.     

Mycoplasma synoviae in a released pen-raised wild turkey.

Mycoplasma synoviae (MS) was isolated from the sinus of an adult female "wild-type" turkey found feeding with backyard chickens at a private residence in Randolph County, N.C. Clinical signs included sinusitis, dyspnea, emaciation, diarrhea, and nasal discharge. The bird was seropositive for MS and M. gallisepticum (MG) on the rapid plate agglutination test and had titers of 1:160 for MS and 1:20 for MG on the hemagglutination-inhibition test. Isolations of MS and M. gallopavonis were confirmed by the fluorescent antibody test. This case represents the first and only report of MS in a free-ranging "wild-type" turkey in the eastern United States. Behavioral and other evidence suggests that the bird was a released pen-raised turkey.     

Identification of species-specific and interspecies-specific polypeptides of Mycoplasma gallisepticum and Mycoplasma synoviae

Temporal antisera (TA) prepared in susceptible Leg-horn-type chickens against Mycoplasma gallisepticum and M synoviae were evaluated to determine the extent of cross-reactivity in ELISA and hemagglutination inhibition tests. Species-specific and interspecies-specific polypeptides were identified after electrophoretic separation and protein immunoblotting with reference antisera, TA, and a monoclonal antibody specific for M gallisepticum. Mycoplasma gallisepticum antiserum cross-reacted with M synoviae polypeptides in ELISA and TA immunoblots. Two major M synoviae polypeptides (88 and 53 kilodaltons [kD]) cross-reacted with M gallisepticum antisera in TA immunoblots. An M gallisepticum polypeptide of 70 kD cross-reacted with M synoviae in TA immunoblots. In contrast, M gallisepticum and M synoviae reference antisera cross-reacted when immunoblotted with heterologous antigens. A monoclonal antibody specific for M gallisepticum bound to a 69-kD polypeptide in lectin-purified and whole-cell M gallisepticum protein fractions in immunoblot assays. The lectin-purified fraction hemagglutinated chicken RBC. Seemingly, the 69-kD polypeptide may constitute all or part of the M gallisepticum hemagglutinin.     

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.     

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.     

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.     

Ribosomal RNA gene probes to detect intraspecies heterogeneity in Mycoplasma gallisepticum and M. synoviae

Intraspecies genotypic heterogeneity among strains of Mycoplasma gallisepticum and M. synoviae was tested using genomic fingerprints with a ribosomal RNA (rRNA) gene probe. The organism's DNA was digested by a restriction endonuclease, electrophoresed, transferred to a nitrocellulose sheet, and hybridized with 32P-labeled pMC5 plasmid carrying the highly conserved rRNA genes of M. capricolum. The resulting hybridization patterns indicated a degree of genotypic heterogeneity among M. gallisepticum strains more pronounced than among the M. synoviae strains tested. Most importantly, the live vaccine F strain of M. gallisepticum could be distinguished from virulent field isolates of this species, enabling the detection and identification of the F strain in areas in which vaccination with this strain has taken place. Genomic fingerprints with an rRNA gene probe can thus be added to the battery of tools useful in taxonomy at the intraspecies level and in epidemiology of mycoplasmosis in poultry.     

Survey of campylobacter, salmonella and mycoplasmas in house crows (Corvus splendens) in Malaysia

House crows (Corvus splendens) in Selangor, Malaysia were examined for the presence of Campylobacter species, Salmonella species, Mycoplasma gallisepticum and Mycoplasma synoviae by serology, culture and pcr. For the detection of Campylobacter and Salmonella species swabs were taken either from the intestine or cloaca. For the detection of mycoplasmas, swabs were taken either from the choanal cleft or trachea for culture and pcr and serum samples were tested by the rapid serum agglutination (rsa) and monoclonal antibody-blocking elisa (mbelisa) for antibodies to M gallisepticum and M synoviae. For campylobacter, 25.3 per cent of the crows were positive by culture, and the species identified were Campylobacter jejuni and Campylobacter coli. No Salmonella species were isolated. Four of 24 swabs were positive for M gallisepticum dna but none gave positive results for M synoviae dna. No M gallisepticum or M synoviae antibodies were detected by rsa but 60 per cent of the sera gave positive reactions for M gallisepticum and 13 per cent gave positive reactions for M synoviae by mbelisa.     

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%。     

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.     

Laboratory infection of house sparrows (Passer domesticus) with Mycoplasma gallisepticum and Mycoplasma synoviae

House sparrows were infected by aerosol with Mycoplasma gallisepticum (MG) or M. synoviae (MS). MG was reisolated from 5 to 11 sparrows 10 days postinfection, but infection appeared to be temporary. Mycoplasma-free chickens reared in the experimental house became infected with MG during the trial. MS was recovered from only one sparrow. Serological tests were unsatisfactory for diagnosing infected birds. The results suggest that house sparrows may be temporary biological carriers of MG.     

Prevalence of mycoplasma antibodies in lesser prairie-chicken sera

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.     

Recombinant DNA probes for Mycoplasma synoviae

A genomic library was prepared from Mycoplasma synoviae (MS) strain WVU 1853 cloned in plasmid vector pUC8 and transformed in Escherichia coli host JM83. In dot blot assays, four transformed E. coli clones hybridized with 32P-labeled chromosomal DNA of MS but not with 32P-labeled chromosomal DNA of M. gallisepticum (MG) strain S6. In Southern hybridization, each of the CsCl-purified recombinant plasmid clones was shown to contain two MS DNA fragments between 1.0 to 2.3 kbp in length. 32P-Labeled probes prepared from each of the four recombinant plasmids hybridized in dot blot assays with MS strain WVU 1853 and nine MS field isolates but not with MG strains S6, K810, F2F10, four MG field isolates, and 15 other species of avian mycoplasmas.     

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.     

Indirect micro-enzyme-linked immunosorbent assay for the detection of antibodies to Mycoplasma synoviae and M. gallisepticum

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.     

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.     

Nonspecific reactions to Mycoplasma serum plate antigens induced by inactivated poultry disease vaccines

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.