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.     

Airsacculitis in turkeys exposed to Mycoplasma synoviae membranes

In studies to investigate the pathogenesis of mycoplasmal airsacculitis, exudative lesions were produced in turkeys by intra-air-sac inoculation with Mycoplasma synoviae cell membranes and viable organisms. Membrane inocula containing 5 mg of protein produced more severe lesions than inocula containing either 2.5 mg or 1 mg protein. Turkeys exposed to 5 mg of membrane protein developed moderately severe airsacculitis; those exposed to viable organisms developed markedly severe airsacculitis. Microscopic examinations revealed that membrane-induced lesions were generally similar to those resulting from infection but were less severe. At the termination of the study, 8 days after exposure, M. synoviae was isolated from respiratory tract tissues of all turkeys exposed to live organisms, but it was not isolated from any of those exposed to membranes or from unexposed control turkeys. Antibody against M. synoviae was demonstrated with the tube agglutination test in sera from turkeys exposed to membranes and those exposed to live organisms, but it was not demonstrated in sera from unexposed control turkeys.     

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.     

The immunosuppressive effect of Mycoplasma meleagridis on nonreplicating antigens

The humoral responses of Mycoplasma meleagridis-free and -infected turkeys were compared following primary and secondary antigenic stimulation with inactivated Salmonella pullorum or dinitrophenyl-bovine gamma globulin. The mycoplasma-infected groups had significantly lower antibody responses to both antigens. The suppression was more evident in the secondary response. The results suggest that the immune response in turkeys infected with M. meleagridis is similar to that of bursectomized chickens: the ability of chickens to synthesize immunoglobulin G is affected more readily than the ability to synthesize IgM.     

Safety of Mycoplasma gallisepticum vaccine strain 6/85 after backpassage in turkeys

The objective of this research was to evaluate the safety of the 6/85 strain vaccine strain of Mycoplasma gallisepticum in turkeys by backpassing the vaccine strain up to 10 times by contact infection in turkeys and challenging turkeys with the resulting backpassaged strain. The vaccine strain, however, did not spread to in-contact turkeys, and it was necessary to reisolate the organism before challenging turkeys for the next passage. The challenge strain, therefore, was one that had been backpassaged four times in turkeys, with a total in vivo time in turkeys of 66 days. The backpassaged 6/85 vaccine strain was no different in pathogenicity than the original vaccine strain, except that at 10 days postchallenge, it was isolated in higher numbers from air sacs. Both the original 6/85 vaccine strain and the backpassaged strain were apathogenic in turkeys, except for a slightly increased diameter of the tracheal mucosa at 10 days postchallenge; at 20 days postchallenge the tracheal mucosal thickness was no different from that of controls.     

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.     

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.     

Antibody responses of turkeys experimentally exposed to Mycoplasma synoviae.

The antibody response of turkeys exposed to Mycoplasma synoviae intravenously and by way of air sacs was determined by tube agglutination, plate agglutination, hemagglutination-inhibition (HI), and gel diffusion precipitin tests. The results suggested that continued antigenic stimulation was lacking in most turkeys and that the response was due mostly to IgM-type immunoglobulin. Under those conditions, both types of agglutination tests were effective and were more sensitive indicators of exposure than the HI test. The gel diffusion precipitin test was not considered effective under the conditions of this study. HI activity occurred in serums of intravenously exposed turkeys within 4 days of exposure. The sensitivity of this activity to 2-mercaptoethanol treatment suggested that IgM was responsible.     

Virulence of recent isolates of Mycoplasma synoviae in turkeys

Systemic Mycoplasma synoviae (MS) infection was induced experimentally in commercial turkeys with recent MS isolates (K4822D and K4774J) from turkey breeder flocks that exhibited no clinical signs typical of MS infection except for a low incidence of swollen footpads. The virulence of each strain was compared by evaluating gross and microscopic lesions, serologic responses, and MS isolation rates at 10 and 21 days postchallenge and by comparing these results with those obtained from a known virulent isolate (K1968), another previously characterized field isolate (K4463B), and unchallenged controls. All strains induced lesions typical of infectious synovitis but showed distinct differences in the extent of the gross and microscopic lesions and in the isolation rates from the tissues in turkeys. K1968 induced the most extensive lesions in hock and stifle joints and footpads, but strains K4822D, K4774J, and K4463B all induced synovitis and were similar in virulence for synovial tissues. Very mild respiratory lesions were induced by all of the strains studied. All strains yielded strong positive serologic responses. We concluded that these recent field isolates, although able to induce synovitis, are less virulent for turkeys than a known virulent strain. Nevertheless, under severe experimental challenge, these strains have the capability of causing lesions that may be incompatible with economical turkey production.     

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.     

Characterization of a naturally occurring infection of a Mycoplasma gallisepticum house finch-like strain in turkey breeders

An outbreak of Mycoplasma gallisepticum (MG) in commercial turkeys involving very mild clinical signs was difficult to confirm by routine methods. In the first part of this study (trial A), we conducted a bioassay to increase the likelihood of detecting MG. Susceptible turkeys were inoculated with sinus exudates from four different affected commercial turkey flocks. Turkeys were evaluated for clinical signs, as well as by serology and culture of tracheal swabs, at 21 and 42 days postchallenge. An MG isolate from one of the sinus exudates used for inoculation, designated K5054, was very similar to isolates from house finches when characterized by random amplified polymorphic DNA analysis as well as DNA sequence analysis of portions of the phase-variable putative adhesin protein (pvpA) gene, a lipoprotein gene, and the cytadhesin gapA/mgc1 gene. The turkeys inoculated with the K5054 sinus exudate seroconverted in the absence of severe clinical signs. There was a single reisolation of K5054 from these turkeys 42 days postchallenge. Susceptible contact turkeys were commingled with the K5054-inoculated turkeys at 49 days postchallenge. We found no evidence of transmission of MG to the contacts by culture or serology at 7, 21, or 35 days after commingling. In the second part of this study (trial B), we challenged the contacts and K5054 sinus exudate-inoculated turkeys from trial A with virulent R strain 88 days after the K5054 sinus exudate inoculation. On necropsy 10 days postchallenge, the evaluation of gross and microscopic lesions, serology, and culture showed that the turkeys previously inoculated with K5054 sinus exudate were protected against disease and reinfection.     

Comparison of strains of Mycoplasma iowae

Comparison of biochemical test results and protein electrophoretic patterns of 21 strains of Mycoplasma iowae indicated that all were similar. Comparison of agglutination test results indicated marked within-species antigenic variation. None of 21 antigens prepared from different strains were effective in demonstrating turkey antibody against five reference strains. Examination of sera from turkeys exposed by intra-air-sac inoculation to two pathogenic strains also indicated antigenic variation. Neither the M. iowae type-strain, Iowa 695, nor the other reference strains were effective in demonstrating antibody against both strains used to expose the turkeys. These findings suggest that antigenic variation may be a major problem in effective serodiagnosis of M. iowae infections.     

Safety and efficacy of the Mycoplasma synoviae MS-H vaccine in turkeys

The live, attenuated, temperature-sensitive Mycoplasma synoviae (MS) vaccine strain MS-H is used to control virulent MS infection in commercial chicken flocks. However, the safety of this vaccine and its potential to prevent disease in turkeys have not been investigated. In this study, MS-H was shown to colonize the upper respiratory system and to induce an antibody response in turkeys but, even at the maximum release dose, was not found to cause air sac, joint, or tracheal lesions typical of wild-type MS infection. Histopathologic examinations of the vaccinated turkeys after exposure to a virulent MS challenge revealed that administration of the vaccine by aerosol, but not eye drop, at the dose recommended for chickens protected the birds against microscopic lesions and colonization of the virulent MS in trachea. It is concluded that MS-H vaccine is safe for use in turkeys and, when used as aerosol at the dose recommended for commercial chickens, can protect turkeys against tracheal lesions caused by a wild-type MS strain.     

Serological detection of Mycoplasma synoviae infection in turkeys.

The antibody response of turkeys experimentally infected with Mycoplasma synoviae was determined by the serum plate agglutination (SPA), hemagglutination-inhibition (HI), and microagglutination (MA) tests and the enzyme-linked immunosorbent assay (ELISA). No antibody response was detected until 2 weeks postinfection (PI) with the MA test (17% positive), 3 weeks PI with the SPA test (11% positive), 4 weeks with the HI test (21% positive), and 5 weeks PI with the ELISA, and even then, only 16% of the birds were positive. Although at least 89% of the birds were positive by culture, only 58% of the turkeys developed a detectable antibody response.     

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.     

The Attempted Immunization of Turkey Hens with Viable Mycoplasma meleagridis

The attempted immunization of 53 female Broad Breasted White turkeys with five weekly 1.0 ml injections of viable Mycoplasma meleagridis, when combined with insemination with M. meleagridis infected semen, did not produce immunity but only imposed additional infection. This was detrimental to the offspring by increasing the incidence and severity of air sac lesions.     

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.     

Nonspecific reactions to Mycoplasma antigens caused in turkeys sera by Erysipelothrix insidiosa bacterin.

Nonspecific reactions to Mycoplasma meleagridis (MM) and M. gallisepticum antigens were found in the sera of turkeys vaccinated with Erysipelothrix insidiosa (EI) bacterin, but they could be removed by adsorption with EI bacterin. True reactions to MM could not be so removed.     

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.