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.     

Mycoplasma gallisepticum infection in wild-type turkeys living in close contact with domestic fowl

Mycoplasma gallisepticum was isolated from 2 wild-type turkeys (Meleagris gallopavo) and 1 domestic turkey living in close contact on a farm in Tehama County, California. Sinusitis was detected in 2 of 14 wild-type turkeys and in 1 of 12 feral broad-breasted bronze turkeys, but in none of several chickens on the premises. The entire mixed flock was captured, sinus aspirates were collected from affected birds, and blood samples were obtained from all birds for serologic testing. Blood samples also were obtained from 10 domestic turkeys on adjacent premises from which breeding stock had been borrowed. The M gallisepticum isolated from sinus aspirates was typed and inoculated into susceptible chickens, resulting in airsacculitis. California wild turkeys with and without histories of exposure to domestic fowl and wild turkeys shipped into California from Texas for release were tested for antibodies to M gallisepticum, using the plate agglutination test. Evidence of M gallisepticum infection was not found in wild turkeys at any location other than the original premises.     

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.     

Comparative study of Mycoplasma pulmonis and Mycoplasma gallisepticum infections in turkey sinus.

Infraorbital sinuses of young turkeys were injected with virulent strains of Mycoplasma pulmonis and Mycoplasma gallisepticum to compare the diseases caused by the 2 agents. Mycoplasma pulmonis did not cause visible swelling from large quantities of mucous exudate in the sinuses, such as occurs with M gallisepticum, and it could not be recovered by bacteriologic culture technique after 3 weeks. However, slight exudate did accompany the M pulmonis infection. Similarities between the disease caused by M pulmonis and that caused by M gallisepticum included lymphocytic infiltration in the submucosa, swollen epithelial cells, and loss of cilia from sinus epithelial cell surfaces. This strain of M pulmonis, which is pathogenic for rats, was only mildly pathogenic for turkeys and the infection did not persist for long.     

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.     

Vaccination of turkeys against air-sac infection with a temperature-sensitive mutant of Mycoplasma gallisepticum

Turkeys were vaccinated with temperature-sensitive (TS) mutants of Mycoplasma gallisepticum (MG) to determine pathogenicity and immunogenicity. TS 37 was apathogenic yet immunogenic to turkeys, TS 100 was highly pathogenic, and TS 102 was slightly pathogenic and nonimmunogenic. Five or 7 weeks after intranasal vaccination of turkeys with the TS 37 mutant, a highly statistically significant resistance against intra-air-sac challenge with the S6 strain of MG was observed.     

Pathogenic effects on domestic poultry of a mycoplasma gallisepticum strain isolated from a wild house finch

Mycoplasma gallisepticum (MG) has been isolated from wild house finches. The pathogenic effects of MG finch strain (K4058) and MG R-strain were compared after exposure of chickens and turkeys. Gross and histologic lesions, reisolation of the organism, serology, and clinical disease were evaluated. Milder histologic and gross lesions, in addition to lower serologic titers, occurred in birds inoculated with the finch strain. Mortality, concurrent with clinical and gross respiratory signs and lesions, was observed only in chickens challenged with R-strain. Both the MG finch strain and MG R-strain were recovered from the respective challenge groups at 14 and 28 days postexposure. The results show that MG isolated from wild house finches may infect domestic poultry species but causes only mild disease and is less virulent than MG R-strain. Commercial enzyme-linked immunosorbent assay kits best detected the serologic response of chickens and turkeys to the MG finch strain.     

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.     

Molecular variability of house finch Mycoplasma gallisepticum isolates as revealed by sequencing and restriction fragment length polymorphism analysis of the pvpA gene

Mycoplasma gallisepticum, a major pathogen of chickens and turkeys, has caused significant declines in house finch (Carpodacus mexicanus) populations in the eastern United States since it was first observed in this species in 1994. There is evidence that M. gallisepticum infection is now endemic among eastern house finches, although disease prevalence has declined, suggesting an evolving host-parasite relationship. Studies based on randomly amplified polymorphic DNA (RAPD) have documented the presence of a single, unique RAPD profile in house finch M. gallisepticum isolates, suggesting a single point source of origin, which agrees with the known epidemiologic observations. In the present study, we evaluated the molecular variability of 55 house finch isolates as well as 11 chicken and turkey isolates including reference strains of M. gallisepticum. Molecular variability was evaluated by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis and nucleotide sequencing of the pvpA gene, which encodes for the putative cytadhesin protein PvpA. Three different RFLP groups and 16 genotypes were evident from the 55 house finch isolates evaluated. Sequence analysis of pvpA gene PCR products showed that although most house finch M. gallisepticum isolates clustered more closely to each other, others clustered more closely to either turkey or chicken field isolates. These findings suggest that house finch isolates are more polymorphic than previously recognized by RAPD studies. This feature may allow us to learn more about the molecular evolution and epidemiology of this emerging disease host-parasite relationship.     

Pathologic lesions caused by coinfection of Mycoplasma gallisepticum and H3N8 low pathogenic avian influenza virus in chickens

Chickens were infected under experimental conditions with Mycoplasma gallisepticum and low pathogenic avian influenza (LPAI) strain A/mallard/Hungary/19616/07 (H3N8). Two groups of chickens were aerosol challenged with M. gallisepticum strain 1226. Seven days later, one of these groups and one mycoplasma-free group was challenged with LPAI H3N8 virus; one group without challenge remained as negative control. Eight days later, the birds were euthanized and examined for gross pathologic and histologic lesions. The body weight was measured, and the presence of antimycoplasma and antiviral antibodies was tested before the mycoplasma challenge, before the virus challenge, and at the end of the study to confirm both infections. Chickens in the mycoplasma-infected group developed antibodies against M. gallisepticum but not against the influenza virus. Chickens of the group infected with the influenza virus became serologically positive only against the virus, while the birds in the coinfected group developed antibodies against both agents. The LPAI H3N8 virus strain did not cause decrease in body weight and clinical signs, and macroscopic pathological lesions were not present in the chickens. The M. gallisepticum infection caused respiratory signs, airsacculitis, and peritonitis characteristic of mycoplasma infection. However, the clinical signs and pathologic lesions and the reduction in weight gain were much more significant in the group challenged with both M. gallisepticum and LPAI H3N8 virus than in the group challenged with M. gallisepticum alone.     

Experimental infection of turkeys with Mycoplasma gallisepticum of low virulence, transmissibility, and immunogenicity.

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.     

Safety and efficacy of the avirulent Mycoplasma gallisepticum strain K5054 as a live vaccine in poultry

A Mycoplasma gallisepticum (MG) isolate from an atypically mild outbreak in turkey breeders was found to be similar to house finch isolates by DNA analyses. A preliminary study in turkeys showed that this isolate (K5054) caused very mild lesions and protected turkeys against subsequent challenge with a virulent MG strain. In this study, K5054 was further evaluated as a potential vaccine strain in commercial layer-type chickens and turkeys. The safety of K5054 was evaluated by aerosol challenge followed by evaluation of gross and histopathologic lesions as well as serologic reactions and isolation of MG from the trachea and air sacs. Infection of chickens (trial 1) and turkeys (trial 2) with K5054 resulted in little evidence of MG lesions. There was weak seroconversion, and K5054 was consistently reisolated from the tracheas of chickens and turkeys. The efficacy of K5054 as a vaccine was evaluated by aerosol challenge of vaccinated chickens (trial 3) and turkeys (trial 4) with virulent R strain. There was evidence of protection from lesions associated with MG.     

A recombinant PvpA protein-based diagnostic prototype for rapid screening of chicken Mycoplasma gallisepticum infections

Mycoplasma gallisepticum is the primary agent of chronic respiratory disease causing important economic losses in the poultry industry. Serological monitoring is essential to maintain mycoplasma-free breeder flocks and often complicated by the cross-reactions between different mycoplasma species. To overcome serological cross-reactions, a large fragment of the M. gallisepticum PvpA cytadhesin, species-specific surface-exposed protein, was produced in E. coli as a recombinant protein (rPvpA336) and used as a potential diagnostic antigen. The rPvpA336 protein possesses 336 mycoplasma-specific amino acids with relative molecular weight of 44 kDa. A deletion region of 37 amino acids was identified when compared to the wild-type PvpA protein. Immunoreactivity of the rPvpA336 protein has been demonstrated by Western blot analysis with M. gallisepticum-positive and -negative chicken sera. Furthermore, an enzymatic rapid immunofiltration assay (ERIFA) prototype based on the rPvpA336 protein has been developed and its species-specific detection capability has been demonstrated by using M. gallisepticum and/or M. synoviae-positive and -negative chicken sera. In addition to its species-specificity, the ERIFA prototype presents certain advantages such as rapidity, field-applicability and cost-effectiveness. Therefore, these advantages would make the prototype a species-specific rapid diagnostic tool of choice in the field and limited laboratory conditions for screening M. gallisepticum infections.     

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.     

Evaluation of a Mycoplasma gallisepticum strain exhibiting reduced virulence for prevention and control of poultry mycoplasmosis.

Two experiments were conducted to evaluate the virulence and vaccination efficacy of a Mycoplasma gallisepticum (MG) isolate designated MG Intervet 6/85. Virulence of the strain was determined by evaluation of airsacculitis scores following aerosol exposure to the isolate before and after 10 sequential passes in either commercial broiler chickens or commercial turkeys. Two-week-old specific-pathogen-free chickens were vaccinated by aerosol exposure. The birds were challenged with the R' strain of MG at either 4 or 8 weeks post-vaccination. Efficacy was evaluated by airsacculitis scores determined 21 days after challenge. Ten repetitive back-passes of the isolate in chickens and turkeys did not substantially increase the virulence. Virulence for both chickens and turkeys was minimal, while protection elicited by aerosol vaccination in young chickens against virulent R' strain was significant (P less than or equal to 0.05) compared with unvaccinated controls.     

Comparison of Mycoplasma gallisepticum strains and identification of immunogenic integral membrane proteins with Triton X-114 by immunoblotting.

Pooled chicken antisera from 33 and 77 days post Mycoplasma gallisepticum strain R contact-exposure reacted with cell proteins of 19 M. gallisepticum strains. These pooled antisera reacted with more proteins and with greater intensity to reference strains (R, PG31, S6, and A5969) and nine field strains than they did with six other field strains including three (703, 503, and 730) that have been described as serological variants. Following extraction with Triton X-114 the majority of immunogenic M. gallisepticum proteins partitioned exclusively or primarily into the detergent phase indicating that they are integral membrane proteins. This included three immunogenic species-specific proteins (p64, p56 and p26). M. gallisepticum p56 was detected, by immunoblotting, in 18 of 19 strains suggesting that it could serve as an antigen for serological tests. P26 was evident in 13 of 19 strains. Hyperimmune antiserum to p64 reacted with a 64 kDa protein in 19 M. gallisepticum strains, but did not react with seven other avian Mycoplasma spp. There was no evidence found supporting the view that p64 is the hemagglutinin of M. gallisepticum.     

Immunogenicity of Mycoplasma gallisepticum

The serological response and protective immunity elicited in the chicken by the pathogenic Ap3AS strain and the moderately pathogenic 80083 strain of Mycoplasma gallisepticum and variants of strain 80083 attenuated by repeated passage in mycoplasma broth were investigated. Strain 80083 elicited a substantial serum antibody response after administration either in drinking water or by conjunctival sac instillation to 7-week-old SPF chickens. No vaccinated chickens developed air sac lesions when challenged by intra-abdominal (IA) injection with the virulent Ap3AS strain. Chickens vaccinated with strain 80083M (50 broth passages) showed only a weak serological response but were substantially protected when challenged 4 weeks after vaccination. Chickens vaccinated with 80083H (100 broth passages) were serologically negative 4 weeks after vaccination and developed severe air sac lesions after challenge. Thirty-seven-week-old hens vaccinated 6 months previously with strain 80083 had high serum antibody levels and were completely protected against IA challenge with the homologous strain. However, 4/6 showed mild air sac lesions when challenged intra-abdominally with strain Ap3AS. Another group showed high M. gallisepticum serum antibody levels 6 months after vaccination with strain Ap3AS but 4/6 and 2/6 showed mild lesions after IA challenge with strains Ap3AS or 80083, respectively. Strains 80083 or 80083M were administered by conjunctival sac instillation to susceptible 11-week-old commercial pullets at the time of fowl pox vaccination. The concurrent use of both vaccines had no apparent adverse effect on the health of the chickens. Similar protection against IA challenge with strain Ap3AS was produced with the M. gallisepticum vaccines whether used alone or in combination with fowl pox.     

Experimental infection of chickens and turkeys with Mycoplasma gallisepticum reference strain S6 and North Carolina field isolate RAPD type B

During an epidemic of mycoplasmosis in chicken and turkey flocks in North Carolina between 1999 and 2001, isolates of Mycoplasma gallisepticum (MG) from affected flocks were characterized by random amplification of polymorphic DNA (RAPD), and eight distinct RAPD types were identified. MG RAPD type B accounted for more than 90% of the isolates and was associated with moderate-to-severe clinical signs and mortality. The virulence of MG RAPD type B for chickens and turkeys was compared with sham-inoculated negative controls and MG S6 (a virulent strain)-inoculated positive controls. Clinical signs occurred in chickens and turkeys inoculated with either MG RAPD type B or MG S6. However, they were not as frequent or severe as those seen in naturally affected flocks, and there was no mortality in the experimental groups. Based on gross and microscopic findings, MG RAPD type B was equal to or more virulent than MG S6. All MG-inoculated birds were culture and PCR positive at 7 and 14 days postinoculation (PI). Among serological tests, the serum plate agglutination test was positive for the majority of chickens and turkeys (58%-100%) infected with either strain of MG at both 7 and 14 days PI. The hemagglutination inhibition test was negative for all birds at 7 days PI and positive for a few chickens (8%-17%) and several turkey sera (40%-60%) at 14 days PI. Only a single serum was positive by enzyme-linked immunosorbent assay (an MG S6-infected turkey) at 14 days PI.     

The isolation and attenuation of a virus causing rhinotracheitis in turkeys in South Africa

In March 1978, a number of turkeys with severe respiratory symptoms affecting over 80% of the flock were investigated for a possible causative agent. With the standard techniques used for the isolation of bacteriae, mycoplasmae and viruses, only Mycoplasma gallisepticum, Mycoplasma meleagridis and Newcastle disease virus were isolated. Tracheal organ cultures were subsequently prepared from 27-day-old turkey embryos and inoculated with sinus exudate from affected turkeys. After an incubation period of 4 days a virus was isolated with which the typical symptoms, as observed in the field, could be reproduced in susceptible turkeys after 3-5 days. Following primary isolation in tracheal organ cultures, the virus grew readily in embryonated eggs and Vero cells. With the electron microscope, virus-like particles, varying in size from 40 nm-500 nm, were observed, having a pleomorphic shape and studded with fine surface projections. The virus seems to fall into the family Paramyxoviridae. A vaccine produced from attenuated virus in embryonated eggs afforded good protection against mortalities due to airsacculitis that normally follows on to turkey rhinotracheitis infection. The serological and clinical effects of the virus on chickens are also reported on.