Attenuated Phenotype of a Recent House Finch-Associated Mycoplasma gallisepticum Isolate in Domestic Poultry.

Mycoplasma gallisepticum, known primarily as a respiratory pathogen of domestic poultry, has emerged since 1994 as a significant pathogen of the house finch (Haemorhousmexicanus) causing severe conjunctivitis and mortality. House finch-associated M. gallisepticum (HFMG) spread rapidly and increased in virulence for the finch host in the eastern United States. In the current study, we assessed virulence in domestic poultry with two temporally distant, and yet geographically consistent, HFMG isolates which differ in virulence for house finches-Virginia 1994 (VA1994), the index isolate of the epidemic, and Virginia 2013 (VA2013), a recent isolate of increased house finch virulence. Here we report a significant difference between VA1994 and VA2013 in their levels of virulence for chickens; notably, this difference correlated inversely to the difference in their levels of virulence for house finches. VA1994, while moderately virulent in house finches, displayed significant virulence in the chicken respiratory tract. VA2013, while highly virulent in the house finch, was significantly attenuated in chickens relative to VA1994, displaying less-severe pathological lesions in, and reduced bacterial recovery from, the respiratory tract. Overall, these data indicate that a recent isolate of HFMG is greatly attenuated in the chicken host relative to the index isolate, notably demonstrating a virulence phenotype in chickens inversely related to that in the finch host.

Mycoplasma corogypsi-associated polyarthritis and tenosynovitis in black vultures (Coragyps atratus).

Three wild American black vultures (Coragyps atratus) were presented to rehabilitation centers with swelling of multiple joints, including elbows, stifles, hocks, and carpal joints, and of the gastrocnemius tendons. Cytological examination of the joint fluid exudate indicated heterophilic arthritis. Radiographic examination in 2 vultures demonstrated periarticular soft tissue swelling in both birds and irregular articular surfaces with subchondral bone erosion in both elbows in 1 bird. Prolonged antibiotic therapy administered in 2 birds did not improve the clinical signs. Necropsy and histological examination demonstrated a chronic lymphoplasmacytic arthritis involving multiple joints and gastrocnemius tenosynovitis. Articular lesions varied in severity and ranged from moderate synovitis and cartilage erosion and fibrillation to severe synovitis, diffuse cartilage ulceration, subchondral bone loss and/or sclerosis, pannus, synovial cysts, and epiphyseal osteomyelitis. No walled bacteria were observed or isolated from the joints. However, mycoplasmas polymerase chain reactions were positive in at least 1 affected joint from each bird. Mycoplasmas were isolated from joints of 1 vulture that did not receive antibiotic therapy. Sequencing of 16S rRNA gene amplicons from joint samples and the mycoplasma isolate identified Mycoplasma corogypsi in 2 vultures and was suggestive in the third vulture. Mycoplasma corogypsi identification was confirmed by sequencing the 16S-23S intergenic spacer region of mycoplasma isolates. This report provides further evidence that M. corogypsi is a likely cause of arthritis and tenosynovitis in American black vultures. Cases of arthritis and tenosynovitis in New World vultures should be investigated for presence of Mycoplasma spp, especially M. corogypsi.

Extensive variation in surface lipoprotein gene content and genomic changes associated with virulence during evolution of a novel North American house finch epizootic strain of Mycoplasma gallisepticum.

Mycoplasma gallisepticum, a significant respiratory and reproductive pathogen of domestic poultry, has since 1994 been recognized as an emergent pathogen of the American house finch (Carpodacus mexicanus). Epizootic spread and pathognomonic characteristics of house finch-associated Mycoplasma gallisepticum (HFMG) have been studied as a model of an emergent to endemic pathogen in a novel host. Here we present comparative analysis of eight HFMG genomes, including one from an index isolate and seven isolates separated spatially and temporally (1994-2008) across the epizootic, and notably having differences in virulence. HFMG represented a monophyletic clade relative to sequenced poultry isolates, with genomic changes indicating a novel M. gallisepticum lineage and including unique deletions of coding sequence. Though most of the HFMG genome was highly conserved among isolates, genetic distances correlated with temporal-spatial distance from the index. The most dramatic genomic differences among HFMG involved phase-variable and immunodominant VlhA lipoprotein genes, including those variable in presence and genomic location. Other genomic differences included tandem copy number variation of a 5 kbp repeat, changes in and adjacent to the clustered regularly interspaced short palindromic repeats, and small-scale changes affecting coding potential and association of genes with virulence. Divergence of monophyletic isolates from similar time/space in the epizootic indicated local diversification of distinct HFMG sublineages. Overall, these data identify candidate virulence genes and reveal the importance of phase-variable lipoproteins during the evolution of M. gallisepticum during its emergence and dissemination in a novel host in nature, likely mediating an important role at the interface between pathogen virulence and host immunity.

Common garden experiment reveals pathogen isolate but no host genetic diversity effect on the dynamics of an emerging wildlife disease.

Host genetic diversity can mediate pathogen resistance within and among populations. Here we test whether the lower prevalence of Mycoplasmal conjunctivitis in native North American house finch populations results from greater resistance to the causative agent, Mycoplasma gallisepticum (MG), than introduced, recently-bottlenecked populations that lack genetic diversity. In a common garden experiment, we challenged wild-caught western (native) and eastern (introduced) North American finches with a representative eastern or western MG isolate. Although introduced finches in our study had lower neutral genetic diversity than native finches, we found no support for a population-level genetic diversity effect on host resistance. Instead we detected strong support for isolate differences: the MG isolate circulating in western house finch populations produced lower virulence, but higher pathogen loads, in both native and introduced hosts. Our results indicate that contemporary differences in host genetic diversity likely do not explain the lower conjunctivitis prevalence in native house finches, but isolate-level differences in virulence may play an important role.

Molecular characterization of Mycoplasma gallisepticum isolates from turkeys.

Mycoplasma gallisepticum was isolated from several turkey flocks at different locations in the United States that were clinically affected with respiratory disease. Five of these isolates from four series of outbreaks had patterns similar to the 6/85 vaccine strain of M. gallisepticum by random amplified polymorphic DNA (RAPD) analysis using three different primer sets, whereas with a fourth primer set (OPA13 and OPA14), only two of the isolates were similar to 6/85. Results obtained by sequencing portions of the pvpA, gapA, and mgc2 genes and an uncharacterized surface lipoprotein gene indicated that the field isolates had DNA sequences that ranged from 97.6% to 100%, similar to the 6/85 results. In some of the outbreaks there was an indirect association with the presence of commercial layers in the area that had been vaccinated with this vaccine strain, but there was no known close association with vaccinated birds in any of the outbreaks. Turkeys were challenged with two of the field isolates and with 6/85 vaccine strain. Turkeys challenged with the field isolates developed respiratory disease with airsacculitis and a typical M. gallisepticum antibody response, whereas birds challenged with 6/85 developed no respiratory signs or lesions and developed only a weak antibody response. Although these isolates were very similar to the 6/85 vaccine strain, it was not possible to prove that they originated from the vaccine strain-it is possible that they could be naturally occurring field isolates.

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.

Enhancement of enteropathogenic Escherichia coli pathogenicity in young turkeys by concurrent turkey coronavirus infection.

In a previous study, turkey coronavirus (TCV) and enteropathogenic Escherichia coli (EPEC) were shown to synergistically interact in young turkeys coinfected with these agents. In that study, inapparent or mild disease was observed in turkeys inoculated with only TCV or EPEC, whereas severe growth depression and high mortality were observed in dually inoculated turkeys. The purpose of the present study was to further evaluate the pathogenesis of combined TCV/EPEC infection in young turkeys and determine the role of these agents in the observed synergistic interaction. Experiments were conducted to determine 1) effect of EPEC dose, with and without concurrent TCV infection, and 2) effect of TCV exposure, before and after EPEC exposure, on development of clinical disease. Additionally, the effect of combined infection on TCV and EPEC shedding was determined. No clinical sign of disease and no attaching and effacing (AE) lesions characteristic of EPEC were observed in turkeys inoculated with only EPEC isolate R98/5, even when turkeys were inoculated with 10(10) colony forming units (CFU) EPEC (high dose exposure). Only mild growth depression was observed in turkeys inoculated with only TCV; however, turkeys inoculated with both TCV and 10(4) CFU EPEC (low dose exposure) developed severe disease characterized by high mortality, marked growth depression, and AE lesions. Inoculation of turkeys with TCV 7 days prior to EPEC inoculation produced more severe disease (numerically greater mortality, significantly lower survival probability [P < 0.05], increased frequency of AE lesions) than that observed in turkeys inoculated with EPEC prior to TCV or simultaneously inoculated with these agents. Coinfection of turkeys with TCV and EPEC resulted in significantly increased (P < 0.05) shedding of EPEC, but not TCV, in intestinal contents of turkeys. These findings indicate that TCV infection predisposes young turkeys to secondary EPEC infection and potentiates the expression of EPEC pathogenicity in young turkeys.

Prevalence of enteropathogenic Escherichia coli in naturally occurring cases of poult enteritis-mortality syndrome.

Enteropathogenic Escherichia coli (EPEC) previously were identified in poult enteritis-mortality syndrome (PEMS)-affected turkeys and associated as a cause of this disease. In the present study, the prevalence of EPEC in PEMS-affected turkeys was examined retrospectively with archived tissues and intestinal contents collected from 12 PEMS-affected turkey flocks in 1998. Formalin-fixed intestinal tissues were examined by light and electron microscopy for attaching and effacing (AE) lesions characteristic of EPEC, and frozen (-75 C) intestinal contents were examined for presence of EPEC. Escherichia coli isolates were characterized on the basis of epithelial cell attachment, fluorescent actin staining (FAS) test, and presence of E. coli attaching/effacing (EAE), shigalike toxin (SLT) type I, SLT II, and bundle-forming pilus (BFP) genes by polymerase chain reaction procedures. EPEC isolates were examined for pathogenicity and ability to induce AE lesions in experimentally inoculated young turkeys. AE lesions were identified by light microscopy in Giemsa-stained intestines from 7 of 12 PEMS-affected turkey flocks. Lesions consisted of bacterial microcolonies attached to epithelial surfaces with epithelial degeneration at sites of attachment and inflammatory infiltration of the lamina propria. Electron microscopy confirmed the identity of AE lesions in six of seven flocks determined to have AE lesions by light microscopy. EPEC were identified in 4 of 12 flocks on the basis of the presence of EAE genes a nd absence of SLT I and SLT II genes; all isolates lacked BFP genes. EPEC isolates produced AE lesions and variable mortality in turkeys coinfected with turkey coronavirus. In total, EPEC were associated with 10 of 12 (83%) naturally occurring PEMS cases on the basis of identification of AE lesions and/or EPEC isolates. These findings provide additional evidence suggesting a possible role for EPEC in the pathogenesis of PEMS.

Mycoplasmosis in captive crows and robins from Minnesota.

Mycoplasma sturni is a recently described organism previously associated with conjunctivitis in European starlings (Sturnus vulgaris), northern mockingbirds (Mimus polyglottos) and blue jays (Cyanocitta cristata). Herein we describe the isolation of M. sturni from an American crow (Corvus brachyrhynchos) presenting with conjunctivitis. A nested-PCR was designed for identification of M. sturni in clinical specimens and the sensitivity of the reaction was found to be 10 colony-changing units. The organism was found in asymptomatic American crows caged with a nestmate of the crow with conjunctivitis. Mycoplasma sturni also was found in asymptomatic American robins (Turdus migratorius) and in a European starling (Sturnus vulgaris) housed at the same facility as the crows. Heterogenity of M. sturni isolates from different host species was found by random amplified polymorphic DNA (RAPD) analyses. Heterogeneity also was found among M. sturni isolates recovered from American crows. We suggest that M. sturni can successfully infect American crows and American robins with or without the presence of clinical disease. Furthermore, we demonstrate that nested-PCR is an effective method for the detection of M. sturni and that substantial genetic heterogeneity exists among natural isolates of this bacterial pathogen.

Mycoplasmosis in evening and pine grosbeaks with conjunctivitis in Quebec.

An outbreak of conjunctivitis affected evening grosbeaks (Coccothraustes vespertinus) and pine grosbeaks (Pinicola enucleator) in Quebec (Canada) during the winter 1998-99. One to 30% of the individuals from these two species were sick at 13 feeding stations. Sick birds were thin and had unilateral or bilateral catarrhal and lymphoplasmacytic conjunctivitis and rhinitis, and mucopurulent infra-orbital sinusitis. Mycoplasmal organisms were isolated in cultures in an affected evening grosbeak and identified as Mycoplasma gallisepticum by direct immunofluorescence. Random amplified polymorphic DNA (RAPD) fingerprinting of this isolate resulted in a banding pattern that was identical to patterns of M. gallisepticum isolates made from similar lesions in house finches (Carpodacus mexicanus) and American gold finches (Carduelis tristis) throughout eastern North America. Mycoplasma gallisepticum was identified by polymerase chain reaction in another evening grosbeak and a pine grosbeak. These observations suggest that the same strain of M. gallisepticum is the likely etiology for the observed disease in evening and pine grosbeaks in Canada and represent an extension of the host-species range for the ongoing epidemic of M. gallisepticum conjunctivitis in eastern North America.

Mycoplasmal conjunctivitis in songbirds from New York.

A field study was conducted to determine the prevalence of conjunctivitis and Mycoplasma gallisepticum (MG) infections in house finches (Carpodacus mexicanus) and other songbirds common to bird feeders in Tompkins County (New York, USA). Eight hundred two individuals of 23 species and nine families of birds were captured and given physical examinations during the 14 mo study beginning in February 1998. Clinical conjunctivitis (eyelid or conjunctival swelling, erythema, and discharge) was observed in 10% (19/196) of house finches examined, and only in the winter months from November to March. Unilateral conjunctivitis was observed in 79% (15/19) of affected house finches; one case developed bilateral disease between 8 and 18 days following initial examination. Conjunctivitis was observed in a similar proportion of males and females sampled, and body condition scores and wing chord lengths were not significantly different between diseased and non-diseased house finches. Mycoplasma gallisepticum was isolated from 76% (13/17) of finches with conjunctivitis and 2% (3/168) of clinically normal house finches sampled during the study. DNA fingerprints of 11 MG isolates using random amplification of polymorphic DNA (RAPD) techniques showed no apparent differences in banding patterns over the course of the study, suggesting persistence of a single MG strain in the study population. The prevalence of conjunctivitis and MG infections declined in house finches between February/March 1998 and February/March 1999 (23% to 6%, and 20% to 5%, respectively), but only the former was significant (P < 0.05). Conjunctivitis was also observed in four American goldfinches (Carduelis tristis) and one purple finch (Carpodacus purpureus). Mycoplasma gallisepticum infection was confirmed in the purple finch, the first documented case of MG-associated conjunctivitis in this species. The purple finch isolate was similar to house finch isolates from the study site by RAPD analysis. Positive plate agglutination (PA) tests were recorded in one other goldfinch and two purple finches, suggesting exposure of these individuals to MG. Positive PA tests were also obtained from two brown-headed cowbirds (Molothrus ater) and four tufted titmice (Parus bicolor), but MG infection could not be confirmed in these cases due to lack of samples. Based on these findings, the prevalence of MG infections in hosts other than house finches appear to be low in the population sampled. There is growing evidence, however, that songbird species other than house finches are susceptible to MG infection and disease.

Mycoplasma sturni from blue jays and northern mockingbirds with conjunctivitis in Florida.

Northern mockingbirds (Mimus polyglottos) and blue jays (Cyanocitta cristata) in a Florida (USA) wildlife care facility developed clinical signs and gross lesions suggestive of the ongoing outbreak of Mycoplasma gallisepticum (MG) conjunctivitis in house finches (Carpodacus mexicanus) and American goldfinches (Carduelis tristis). Mycoplasmal organisms were cultured from conjunctival/corneal swabs of birds with sinusitis, conjunctivitis, and/or epiphora. All of the isolates tested were identified as Mycoplasma sturni by indirect immunofluorescence. Mycoplasma sturni as well as MG should be considered in the differential diagnosis of songbirds with conjunctivitis.

Mycoplasmas in wild turkeys living in association with domestic fowl.

One hundred and nineteen Merriam's wild turkeys (Meleagris gallopavo merriami) and 31 domestic chickens coexisting on a ranch in west-central Colorado (USA) were surveyed for mycoplasmosis by serologic and cultural methods. Although no clinical signs were apparent in any wild turkeys tested, 51 (43%) had positive rapid plate agglutination (RPA) reactions for M. gallisepticum (MG) and/or M. synoviae (MS); 37% of 56 adults and 48% of 63 subadults were classified as positive reactors to MG and/or MS. No turkeys tested in 1992 (n = 61) and 17 (29%) of 58 turkeys tested in 1993 were RPA-positive for M. meleagridis (MM). Hemagglutination inhibition (HI) test results were negative for MG, MS and MM as were most enzyme-linked immunosorbent assay (ELISA) test reactions (MG = 99%, MS = 93%, MM = 87%). Immunoblotting showed mild to moderate reactivity to MG proteins in 49% of 41 samples tested. Most chickens were strongly positive for MS by RPA (81%), HI (58%) and ELISA (87%); 48% also were positive for MG by RPA but all were MG-negative by HI and ELISA. No pathogenic mycoplasmas were isolated from either group of birds. Mycoplasma gallopavonis was commonly identified from the wild turkeys, and M. gallinaceum was isolated from both the chickens and wild turkeys. In a transmission study conducted in 1994, disease-free domestic turkeys failed to seroconvert when co-housed with wild turkeys from this population that were RPA-positive for MG. Collectively, the results of this study were inconclusive regarding the status of pathogenic mycoplasmas within this wild turkey population.

Mycoplasma synoviae has two distinct phase-variable major membrane antigens, one of which is a putative hemagglutinin.

Mycoplasma synoviae is a major pathogen of poultry, causing synovitis and respiratory infection. A cluster of 45- to 50-kDa membrane proteins is immunodominant in strain WVU-1853. Four distinct proteins were identified in this cluster by high-pressure liquid chromatography. Monoclonal antibodies and monospecific antisera against each established that they fell into two groups, MSPA and MSPB, each containing two members distinguishable by a difference in hydrophobicity. A 25- to 30-kDa membrane protein (MSPC) was shown to be antigenically related to the MSPB proteins. Considerable variation in the size and expression of MSPA and MSPB was observed among different strains of M. synoviae. Examination of expression in colonies of strain WVU-1853 established that both MSPA and MSPB (and MSPC) were phase variable. Immunostaining of MSPB (and MSPC) with monoclonal antibodies exhibited quantal variation, with three distinct levels observed between and within colonies. Hemadsorption by M. synoviae colonies was also found to be phase variable, with some colonies exhibiting sectorial expression of hemadsorption. Monospecific antisera against MSPA inhibited hemagglutination, but neither monoclonal antibodies nor monospecific antisera against MSPB could inhibit hemagglutination. However, loss of the capacity to hemadsorb by individual clones was associated with loss of expression of both MSPA and MSPB. These findings have elucidated the complexity of structure, function, and expression of the 45- to 50-kDa membrane protein cluster of M. synoviae, and they suggest that all members of the cluster may be involved in adhesion.

Molecular epidemiologic investigations of Mycoplasma gallisepticum conjunctivitis in songbirds by random amplified polymorphic DNA analyses.

An ongoing outbreak of conjunctivitis in free-ranging house finches (Carpodacus mexicanus) began in 1994 in the eastern United States. Bacterial organisms identified as Mycoplasma gallisepticum (MG) were isolated from lesions of infected birds. MG was also isolated from a blue jay (Cyanocitta cristata) that contracted conjunctivitis after being housed in a cage previously occupied by house finches with conjunctivitis, and from free-ranging American goldfinches (Carduelis tristis) in North Carolina in 1996. To investigate the molecular epidemiology of this outbreak, we produced DNA fingerprints of MG isolates by random amplification of polymorphic DNA (RAPD). We compared MG isolates from songbirds examined from 1994 through 1996 in 11 states, representing three host species, with vaccine and reference strains and with contemporary MG isolates from commercial poultry. All MG isolates from songbirds had RAPD banding patterns identical to each other but different from other strains and isolates tested. These results indicate that the outbreak of MG in songbirds is caused by the same strain, which suggests a single source; the outbreak is not caused by the vaccine or reference strains analyzed; and MG infection has not been shared between songbirds and commercial poultry.

Transmissibility of live Mycoplasma gallisepticum vaccine strains ts-11 and 6/85 from vaccinated layer pullets to sentinel poultry.

In separate trials, layer pullets were vaccinated with Mycoplasma gallisepticum (MG) strain 6/85 or strain ts-11 commercially produced live vaccines. For a 15-wk postvaccination (PV) period, vaccinates were commingled with unvaccinated pullets and were in indirect contact with sentinel groups of pullets, broiler breeders, turkey breeders, or meat turkeys in adjoining pens. Infectivity and transmissibility of vaccine strains were determined by tracheal culture and serology at 1 wk followed by 3-wk intervals PV. Strain 6/85 was recovered from 0%-20% of vaccinates, but not from commingled pullets or sentinel birds. Strain ts-11 was recovered from 60%-90% of vaccinates and 0%-40% of commingled pullets but not from any of the sentinel birds. No birds in the 6/85 vaccine trial tested positive for MG antibodies by serology. MG enzyme-linked immunosorbent assays detected positive responses in ts-11 vaccinates (range = 10%-70%) at 42, 63, 84, and 105 days PV, and commingled pullets (10%) at 84 and 105 days PV. MG serum plate agglutination tests detected positive responses in 90% and 20% of ts-11 vaccinates at 42 and 105 days PV, respectively, and commingled pullets (10%) at day 42 PV. Clinical signs, morbidity, or mortality suggestive of pathogenic MG infection were not observed in any bird during either trial, and no gross lesions were observed at necropsy. Random amplified polymorphic DNA analysis was capable of distinguishing each of the vaccinal strains 6/85 and ts-11 from each other by their distinct DNA banding patterns.

Antibody responses of chickens to inoculation with Mycoplasma gallisepticum membrane proteins in immunostimulating complexes.

Membrane proteins of Mycoplasma gallisepticum (MG) strain R were extracted with the detergent Mega-10 and incorporated into immunostimulating complexes (ISCOMs). A membrane protein of approximately 64 kD (p64) molecular weight was a major component of MG ISCOMs. Six-week-old specific-pathogen-free leghorn chickens were inoculated by various routes (subcutaneous; combined intranasal and eyedrop; and combined subcutaneous, intranasal, and eyedrop) with 10 micrograms MG proteins in ISCOMs, or inoculated subcutaneously with 10 micrograms MG proteins in Freund's adjuvant. Subcutaneous inoculation of MG ISCOMs, or MG Freund's adjuvant resulted in higher sero-positive rates (detected by enzyme-linked immunosorbent assay) in serum and respiratory tract washings, compared to combined routes of MG ISCOM inoculation. In chickens inoculated subcutaneously with MG ISCOMs antibodies were first detected at 31 days postinoculation (PI) and the sero-positive rate peaked at 56 days PI. Sero-positive rates started to decline at day 64 PI. In the Freund's adjuvant group, MG antibodies were first detected at day 21 PI, and the sero-positive rate peaked at day 39 PI and did not decline. MG antibodies were detected by ELISA in upper respiratory tract and tracheal washes from chickens inoculated subcutaneously with MG ISCOMs and MG Freund's adjuvant. Immunoblots to MG strain R whole cell proteins showed that respiratory tract washings and sera from chickens inoculated subcutaneously with MG ISCOMs contained immunoglobulins to MG proteins, with a prominent reaction to p64.

Mycoplasma gallisepticum isolated from house finches (Carpodacus mexicanus) with conjunctivitis.

An epornitic of conjunctivitis in free-flying house finches (Carpodacus mexicanus) occurred in several mid-Atlantic and eastern states of the USA in 1994. Clinical signs and gross lesions ranged from mild to severe unilateral or bilateral conjunctival swelling with serous to mucopurulent drainage and nasal exudate. Microscopic lesions consisted of chronic lymphoplasmacytic conjunctivitis, rhinitis, and sinusitis. Notably slow-growing mycoplasmas were isolated from conjunctival and/or infraorbital sinus swabs from clinically affected birds. Isolates were identified as Mycoplasma gallisepticum (MG) by direct immunofluorescence and DNA probe-based polymerase chain reactions. These findings suggest that MG is the likely etiology for this epornitic of conjunctivitis in house finches.

Identification of Mycoplasma synoviae immunogenic surface proteins and their potential use as antigens in the enzyme-linked immunosorbent assay.

Specific immunogenic proteins of Mycoplasma synoviae (MS) strain WVU-1853 were purified and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western transfer using anti-MS, anti-M. gallisepticum, and non-immune chicken sera. A cluster of prominent immunoreactive proteins with molecular masses from 46 to 52 kDa (p46-52) and less reactive single proteins of approximately 22 and 92 kDa were shown to partition into the detergent phase of Triton X-114 MS lysates, suggesting that these amphiphilic polypeptides are integral membrane proteins. Monoclonal antibodies, produced by immunizing mice with MS whole cell proteins and shown to bind species-specific determinants, reacted strongly with p46-52 and less intensely with the 22-kDa protein, but they did not react with the 92-kDa protein. A protein fraction extracted from the Triton X-114 detergent phase and further purified by ion-exchange chromatography was found to be highly enriched in p46-52 and was used as antigen in a prototype enzyme-linked immunosorbent assay (ELISA) to detect MS antibodies. Seventy serum samples were taken variously from specific-pathogen-free chickens experimentally infected with MS or MG and from commercial broiler breeder chickens vaccinated for MS and MG. All samples were tested by both rapid serum agglutination and a prototype ELISA described herein; the two tests were strongly correlated (r = 0.776). The results indicate that the ELISA antigen used--the immunodominant cell surface proteins in the p46-52 cluster--appears to be a good candidate for use in the development of improved rapid diagnostics of MS infections in birds.

Protective immune response to Mycoplasma gallisepticum demonstrated in respiratory-tract washings from M. gallisepticum-infected chickens.

Chickens inoculated with Mycoplasma gallisepticum (MG) produced IgA, IgM, and IgG detectable in washings from the upper respiratory tract (URTW; nasal sinuses and turbinates) and lower respiratory tract (LRTW; trachea, lungs, and air sacs). URTW and LRTW from infected chickens had significant protective effects in a MG-inoculated tracheal-ring-organ-culture system. Protective effects in vitro correlated positively with total MG-specific immunoglobulin titer, but not IgA titer, as determined by enzyme-linked immunosorbent assay. URTW and LRTW from infected chickens inhibited attachment of MG to tracheal-ring-organ cultures in a dose-dependent manner. This suggests that chickens produce a protective immune response to MG that locates in the respiratory tract and that attachment inhibition may be responsible for this protective effect.