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.

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.

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.

Inhibition of Mycoplasma gallisepticum growth and attachment to chick tracheal rings by antibodies to a 64-kilodalton membrane protein of M. gallisepticum.

A Mycoplasma gallisepticum (MG) strain R protein of 64 kilodaltons (p64) was partially digested from the surface of the bacterium by trypsin. Monospecific polyclonal anti-p64 IgG inhibited attachment of MG to chick tracheal rings by as much as 69%. However, trypsin treatment of viable MG cells did not reduce attachment to tracheal rings or hemagglutination titer. Anti-p64 IgG inhibited growth of MG strain R in broth and on solid media, inhibited the uptake of radiolabeled thymidine, but did not inhibit hemagglutination. Anti-p64 IgG inhibited growth of eight MG strains on solid medium. The degree of growth inhibition varied widely depending on the strain and correlated positively with the reported virulence of the MG strains with one exception (A5969). An IgG monoclonal antibody to p64 (MyG 001) inhibited growth of MG strain R on solid and in broth media. The strong attachment-inhibition activity of anti-p64 IgG may result from its growth-inhibiting activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of MG strains suggested that p64 is expressed in higher amounts in vitro in virulent strains (R, S6) than in strains of low virulence (F, M876, K503, K703, K730). P64 should be used to immunize chickens to determine if it can stimulate a growth and attachment-inhibiting response in the respiratory tract.

An outbreak of Mycoplasma synoviae infection in North Carolina turkeys: comparison of isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and restriction endonuclease analysis.

Mycoplasma synoviae (MS) isolates made in 1988-89 from turkey flocks in North Carolina, Missouri, and Ontario, Canada, were compared with each other and MS reference strains (WVU-1853, F10-2AS, Neb-3S, and K1968) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of cell proteins and restriction endonuclease analysis (REA) of DNA. SDS-PAGE and REA indicated considerable homology among MS reference strains and recent field isolates. However, sufficient differences were resolved to identify the MS reference strains as different from each other and the field isolates, and to classify seven of nine recent field isolates as a cluster of nearly identical strains. The results suggest that flocks infected with members of the cluster were epizootiologically associated, possibly by a common or point source of infection.

The pathogenesis of infectious bursal disease: serologic, histopathologic, and clinical chemical observations.

Infectious bursal disease in 35-day-old specific-pathogen-free (SPF) chickens was characterized clinically by its acute onset and brief duration. Clinical signs included depression, anorexia, diarrhea, and polyuria. A detectable precipitin antibody response occurred between 3 and 5 days postinoculation. Evaluation of pooled serum samples obtained from infectious bursal disease virus (IBDV)-infected chickens revealed transient changes in potassium, cholesterol, uric acid, lactate dehydrogenase, serum glutamic-oxalic transaminase, and serum proteins. Individual serum samples analyzed for uric acid concentration indicated that several IBDV-infected chickens had serum uric acid concentrations above the normal comparison range. Histopathologic examination of lymphoid and nonlymphoid tissues from IBDV-infected SPF chickens affirmed that the predominant lesion was lymphoid necrosis in the bursa of Fabricius. Other lymphoid organs were much less severely affected and possessed greater regenerative potential. Nonspecific and relatively mild changes were found in the liver and kidney: hepatic lipidosis and necrosis, renal intratubular crystalline deposits (probably urates), and increased ectopic lymphoid foci. There was no evidence of immune-complex-mediated arteritis/vasculitis in the sartorius muscle or any other tissue examined. Histopathologic and ultrastructural evidence of glomerulonephritis was rare but compatible with acute immune complexemia.

Immune-complex involvement in the pathogenesis of infectious bursal disease virus in chickens.

Frozen kidney sections from chickens inoculated with infectious bursal disease virus (IBDV) were stained with fluorescein-conjugated rabbit anti-chicken gamma-globulin. Fluorescence was observed in the renal glomeruli of infected chickens, indicating that gamma-globulins, probably in the form of immune complexes, had lodged in the glomeruli of IBDV-infected chickens. This suggests that immune complexes may play an important role in the pathogenesis of IBDV infections in chickens.

Pasteurellosis in bobwhite quail.

A flock of 5000 six-week-old bobwhite quails (Colinus virginianus) experienced high mortality (52%) over a 2-day period. Mortality was 99% within a 6-day period. Clinical signs were depression followed shortly by death. Gross lesions observed in dead quails were congested lungs and, in a few cases, mottled livers. Histopathologic examination revealed severe, diffuse, heterophilic interstitial pneumonia and multifocal areas of hepatic and splenic necrosis with numerous intracellular and extracellular short bacterial rods. Serotype 3, 4, 15, 16, Pasteurella multocida, isolated from the index case, caused 50% mortality in experimentally inoculated bobwhite quails within 9 to 24 hours. This report indicates that pasteurellosis can cause peracute disease in bobwhite quails with very high mortality.

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.

Clinical Mycoplasma gallisepticum infection in multiplier breeder and meat turkeys caused by F strain: identification by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, restriction endonuclease analysis, and the polymerase chain reaction.

In February 1991, a flock of North Carolina multiplier breeder turkeys experienced respiratory signs, sinusitis, airsacculitis, and increased mortality. Mycoplasma gallisepticum (MG) was isolated, and appropriate control measures were initiated. Ultimately, this outbreak involved several breeder flocks of an integrated turkey production company before the last infected flock was identified in May 1991. During this time, MG was also isolated from a flock of commercial layer-type chickens raised as pullets in close proximity to the index turkey flock. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and restriction endonuclease analysis indicated that these isolates were identical to each other and to examples of the vaccinal F strain. Additionally, MG isolates from the affected turkey breeder and layer flocks were identified as MG F strain by use of an F strain-specific DNA probe and polymerase chain reaction. A separate outbreak of MG disease in several meat-turkey flocks of a Midwest producer/processor yielded isolates identified as F strain by the polymerase chain reaction. These studies demonstrated: 1) the utility of newer technologies for disease outbreak investigations; and 2) the potential of MG F strain to cause disease in breeder and meat turkeys under field conditions.

Humoral immune response of turkeys to strain S6 and a variant Mycoplasma gallisepticum studied by immunoblotting.

Two putative variant Mycoplasma gallisepticum (MG) strains (M876 and M35), originally isolated from commercial turkeys, were compared with eight well-characterized MG strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE protein profiles indicated that the variant strains were correctly classified as MG based on homologous patterns in species-specific regions of the electrophoretic profiles. However, differences in protein profiles also indicated that variant strains M876 and M35 were different from each other and the other MG strains tested. Immunoblotting was used to assess the humoral immune response of turkeys to infection with the S6 reference strain or M876 variant strain of MG. Immunoblots using antisera to M876 showed that seroconversion to this isolate was slower, and to fewer MG proteins when compared with immunoblots using antisera to S6. Immunoblot analyses further indicated that pooled antisera from turkeys inoculated with either S6 or M876 reacted with each of 10 MG strains tested. However, pooled S6 antisera reacted with greater intensity and with more MG proteins than did pooled M876 antisera. The species-specific immunodominant proteins with the greatest potential for use as antigens in serologic tests appeared to be those of 64 (p64) and 56 (p56) kilodaltons molecular mass.

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.

An infectious bursal disease virus outbreak in 14- and 15-week-old chickens.

Infectious bursal disease virus (IBDV) observed in a flock of 14- and 15-week-old chickens was typical of the acute symptomatic IBDV infections more common in younger birds. High flock morbidity was indicated by a marked decrease in feed consumption, although deaths were not excessive. At necropsy, affected birds had small hemorrhages in thigh muscles, creamy-yellow-colored bursae of Fabricius with prominent longitudinal striations, and swollen mottled kidneys. Histopathologic examination revealed bursal lesions typical of IBDV infection. One of six sera from necropsied birds was positive for antibody to IBDV in the agar-gel precipitin (AGP) test, and one week later all 35 samples tested were positive. Bursae were homogenized and found to contain IBDV as evidenced by precipitation, with antibody to IBDV, in the AGP test.

Cryptosporidia-positive rates of avian necropsy accessions determined by examination of auramine O-stained fecal smears.

Fecal smears from 112 avian necropsy accessions representing 431 birds were stained with auramine O and examined for Cryptosporidium oocysts by fluorescence microscopy. Stained Cryptosporidium oocysts fluoresced bright yellow-green and were easily differentiated from extraneous material by their uniform small size (approx. 5 micron) and morphology. The rates of cryptosporidia-positive accessions were 27.3% (9/33) of broilers, 10% (3/30) of broiler breeders, and 5.9% (1/17) of layers. Further analyses of available data for various risk factors that may have influenced rates of cryptosporidia-positive samples in broilers, broiler breeders, and layers failed to show significant relationships. However, it was apparent that positive samples were clustered within accessions and not scattered throughout the population sampled. This survey also resulted in the first reported identification of Cryptosporidium oocysts from a budgerigar, macaw, and tundra swan.

Staphylococcus-induced gangrenous dermatitis in broilers.

An infectious bursal disease (IBD)-vaccinated flock of 23,900 broilers, 17 days of age, experienced sudden onset of depression, dermatitis, and mortality. Postmortem examination showed extensive subcutaneous serosanguineous fluid accumulation over the pectoral muscles, discrete hepatic whitish foci, fluid-filled intestines, and small, flaccid bursae of Fabricius. Gram-stained impression smears from the affected areas revealed numerous gram-positive cocci. Aerobic culture of liver and subcutaneous tissue consistently produced heavy growth of penicillin-sensitive Staphyloccus aureus. Histopathologically, subcutaneous tissue showed diffuse hemorrhage and large numbers of gram-positive cocci with severe congestion and hemorrhage of the underlying skeletal muscle. Liver sections showed multiple, randomly scattered areas of acute coagulation necrosis with numerous gram-positive cocci. Bursal lesions were characterized by extensive follicular necrosis and collapse. A diagnosis of staphylococcal gangrenous dermatitis secondary to IBD was made. Mortality returned to preinfection levels within 72 hours after penicillin was added to the drinking water.

Experimental reproduction of enteritis in bobwhite quail (Colinus virginianus) with Cryptosporidium and reovirus.

Five-day-old bobwhite quails were inoculated with reovirus and Cryptosporidium previously isolated from the intestinal contents of young, commercially raised bobwhite quails experiencing severe enteritis. Quails inoculated with reovirus alone did not develop clinically apparent disease, infection was localized principally in the intestinal tract, and no lesions were detected. Quails inoculated with Cryptosporidium, alone or with reovirus, developed severe enteritis with high mortality and marked growth depression. Cryptosporidia caused blunting of intestinal villi and provoked a mononuclear cell response in the lamina propria. The severity of intestinal lesions correlated with numbers of parasites. An apparent synergistic effect in dually infected quails was indicated by enhanced Cryptosporidium oocyst shedding, greater numbers of cryptosporidia in the intestinal tracts, and systemic reovirus infection. In addition, multifocal liver necrosis was detected in dually infected quails but was absent in quails infected with only reovirus or Cryptosporidium. The results suggest that Cryptosporidium promoted systemic spread of reovirus, and reovirus intensified Cryptosporidium infection, but no significant synergistic effect on mortality or weight gain was detected. The most important agent in the naturally occurring acute enteritis of bobwhite quails was Cryptosporidium.

Interaction of reovirus and Cryptosporidium baileyi in experimentally infected chickens.

No clinical signs, gross lesions, or increased mortality were observed in specific-pathogen-free chickens orally inoculated at 5 days of age with Cryptosporidium baileyi, reovirus 2035, reovirus 2408, or combinations of these agents. Weight gain of chickens inoculated with only reovirus 2408 was depressed 0-8 days postinoculation (PI) (P less than 0.01) but not for the 21-day period PI. Weight gain of chickens inoculated with only reovirus 2035 was not affected. Cryptosporidium baileyi infection significantly depressed weight gain 8-14 days PI but not for the entire 21-day period PI. Weight gain of chickens infected with both C. baileyi and reovirus 2035 was significantly depressed 0-14 days PI and for the entire 21-day period PI. Dual infection with C. baileyi and either reovirus appeared to promote shedding of both agents. Cryptosporidia were found principally in the rectum 2-10 days PI and in the bursa of Fabricius 6-10 days PI. Reovirus infection did not cause any microscopic lesions and did not modify lesions caused by C. baileyi infection.