Evaluation of the specificity and sensitivity of two commercial enzyme-linked immunosorbent assay kits, the serum plate agglutination test, and the hemagglutination-inhibition test for antibodies formed in response to Mycoplasma gallisepticum

Two commercial enzyme-linked immunosorbent assay (ELISA) kits, seven serum plate agglutination (SPA) antigens, and the hemagglutination-inhibition (HI) test for antibodies to Mycoplasma gallisepticum (MG) were compared for sensitivity and specificity using known MG-positive and MG-negative sera from leghorn chickens. All SPA antigens proved to be highly sensitive when testing MG-positive sera. Laboratory-prepared SPA antigens yielded fewer positive reactions when testing MG-negative sera than commercial SPA antigens. Both MG ELISA kits showed high rates of positive reactions when testing sera from birds given commercial M. synoviae bacterin, fowl coryza (Haemophilus paragallinarum) bacterin, inactivated infectious bursal disease virus vaccine, and to a lesser extent fowl cholera (Pasteurella multocida) bacterin. Immunization with Frey's medium with 12% swine serum-in-oil or Staphylococcus aureus-in-oil resulted in sera which yielded numerous positive ELISA reactions. During the first 1 to 3 weeks, antibodies induced by experimental infection with MG were better detected by the SPA test than by the ELISAs and the HI test, thus confirming the SPA test's importance in Mycoplasma diagnostic serology. The HI test can serve to confirm positive SPA results.     

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

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

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.     

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.     

Use of an avidin-biotin enhanced dot-immunobinding assay to detect antibodies for avian mycoplasma in sera from Iowa market turkeys

A dot-immunobinding assay, amplified with avidin and biotin (DAB assay), was used to detect serum antibodies to Mycoplasma iowae in immunized turkeys. The DAB assay was used to test serum samples from 122 commercial market turkey flocks obtained from four Iowa processing plants. The samples were pooled and tested for the presence of antibodies to four species of Mycoplasma spp. considered to be important pathogens for turkeys: M. gallisepticum (MG), M. iowae (MI), M. meleagridis (MM), and M. synoviae (MS). The occurrence of antibodies against these mycoplasmas, as determined by the DAB assay, were 5.7% for MG, 18.0% for MI, 77.9% for MM, and 9.8% for MS.     

Survey of peafowl (Pavo cristatus) for potential pathogens at three Michigan zoos.

Blood samples collected from 31 free-roaming peafowl from three zoos in Michigan were tested serologically. Antibody titers were present against avian adenovirus and Bordetella avium in 19.3% and 61.3% of the samples, respectively. Serum plate agglutination tests were positive for Mycoplasma meleagridis and Mycoplasma synoviae in 3.2% and 38.7% of the samples, respectively. All birds were seronegative for avian influenza, Newcastle disease virus, West Nile virus, Mycoplasma gallisepticum, Salmonella pullorum, Salmonella typhimurium, and Giardia sp. No parasites were seen in blood smears. Cloacal swabs were cultured for anaerobic, aerobic, and microaerophilic bacteria. Clostridium perfringens type A and Escherichia coli were cultured most frequently from 64.5% and 29% of the samples, respectively, whereas Salmonella sp. and Campylobacter sp. were not isolated. Fecal samples contained moderate numbers of ascarid and Capillaria sp. ova and coccidian oocysts. Female biting lice (Goniodes gigas) were identified on three birds.     

An enzyme-linked immunosorbent assay for the detection of antibodies to Mycoplasma gallisepticum in experimentally infected chickens

An indirect enzyme-linked immunosorbent assay (ELISA) was developed and tested for its ability to detect humoral response to Mycoplasma gallisepticum in chickens. Two antigens were used in the solid phase of the assay. Antigen 1 was a membrane-derived sodium dodecyl sulfate (SDS)-solubilized preparation; Antigen 2 was prepared in the same manner as Antigen 1 but was passed through an immunoadsorbent column containing rabbit anti-medium antibodies. Test conditions were optimized for incubation times and temperatures. Antigen, serum, and enzyme conjugate concentrations were standardized, and reproducibility was determined. A baseline value, representing a positive or negative result, was established independently for both antigens. The assay was then used to detect anti-M. gallisepticum antibodies in experimentally infected chickens. Serum samples collected at 0, 2, 5, 7, 10, 14, 21, 28, and 35 days postinfection (PI) were analyzed by serum plate agglutination (SPA), hemagglutination-inhibition (HI), and ELISA with both Antigens 1 and 2. ELISA was found to be less sensitive but more specific than SPA and more sensitive than HI. The ELISA was more sensitive with Antigen 1 than with Antigen 2. The former assay correctly identified 79% of the serum samples positive for M. gallisepticum by 7 days PI and 100% of the positive birds by 35 days PI. When the absorbance values for each group of birds were averaged, the ELISA successfully identified the M. gallisepticum-infected birds as uniformly positive 7 through 35 days PI and correctly identified all other groups negative for M. gallisepticum through 35 days PI.     

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.     

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.     

Survey of the health and husbandry of small poultry flocks in Great Britain

An investigation into the health and husbandry of 15 small poultry flocks was undertaken. Each flock was visited in July and a questionnaire on management practices and disease history was completed. The flocks were clinically examined and serological tests for Salmonella pullorum, Mycoplasma gallisepticum, M synoviae, M meleagridis, Newcastle disease, infectious bursal disease, infectious bronchitis, eggdrop syndrome 76, adenoviruses and reoviruses were carried out. Oesophageal and cloacal swabs were cultured for mycoplasma and pullorum reactors were cultured. M gallisepticum, M synoviae, M meleagridis and M gallinarum infections were detected and serological reactions for all the viral diseases, except egg drop syndrome 76, were found. Evidence of Newcastle disease and pullorum disease was encountered. Lice were present in five flocks and mites in four flocks. Welfare standards varied.     

Survey of pathogens and blood parasites in free-living passerines.

To determine the disease prevalence of free-living passerines, 1709 passerines were sampled from 38 different field sites in Ohio. Choanal and cloacal swabs were collected from each bird and cultured for the presence of Pasteurella multocida, Salmonella spp., and Escherichia coli by standard microbiologic techniques. In addition, the serum from each bird was analyzed for the presence of antibodies to Mycoplasma gallisepticum, Mycoplasma synoviae, Newcastle disease virus, and avian influenza virus. A blood smear was also made to examine for the presence of blood parasites. Results indicated that the isolation of E. coli varied with bird species, with the European starling having a higher (21.4%) isolation of E. coli. Salmonella spp. were also isolated from these free-living passerines. Pasteurella multocida was not isolated from any of the sampled passerines. These birds did not have antibodies to M. gallisepticum, M. synoviae, Newcastle disease virus, or avian influenza virus. Blood parasites were not detected in any of the birds sampled.     

Health status of northern bobwhite quail (Colinus virginianus) in eastern Kansas

The health status of wild northern bobwhite quail (Colinus virginianus) from Lyon County, Kansas, was evaluated by conducting comprehensive health assessments on 25 birds. Gross lesions indicative of avian pox, ulcerative enteritis, and quail bronchitis were not present. Serologic tests for antibodies to Salmonella pullorum, Salmonella gallinarum, Pasteurella multocida, Mycoplasma gallisepticum, Mycoplasma synoviae, and avian adenoviruses were all negative. Intestinal coccidia (Eimeria spp.) were found in 36% of the birds. Only three species of helminth parasites were found: Dispharynx nasuta in two birds, Cyrnea colini in one bird, and larval Physaloptera sp. in four birds. Arthropod parasites (ticks, lice, mites, and/or chiggers) were present on 96% of the birds examined. Compared with wild bobwhite populations in the southeastern United States, the diversity, prevalence, and intensities of microbial and parasitic agents were low.     

Serologic survey of slaughter-age ostriches (Struthio camelus) for antibodies to selected avian pathogens

Serum samples from 163 slaughter-age ostriches (Struthio camelus) in Ohio and Indiana were tested for antibodies to avian influenza virus (AIV), Newcastle disease virus (NDV), paramyxovirus (PMV) 2, PMV3, PMV7, infectious bursal disease virus (IBDV), Bordetella avium, Mycoplasma synoviae, Mycoplasma gallisepticum, Ornithobacterium rhinotracheale, Salmonella pullorum, Salmonella gallinarum, and Salmonella typhimurium. One ostrich had antibodies to AIV H5N9, 57% of the ostriches had antibodies to NDV, four ostriches had antibodies to both NDV and PMV2, and one ostrich had antibodies to NDV, PMV2, PMV3, and PMV7. None of the ostriches had antibodies to IBDV, B. avium, M. synoviae, M. gallisepticum, O. rhinotracheale, S. pullorum, S. gallinarum, and S. typhimurium. This is the first report of antibodies to avian influenza and PMV7 in ostriches in the United States.     

Breeder turkey hens seropositive and culture-negative for Mycoplasma synoviae.

Four flocks of clinically normal turkey breeder hens were shown to have suspect and positive Mycoplasma synoviae (MS) hemagglutination-inhibition (HI), enzyme-linked immunosorbent assay, and, in some cases, serum plate agglutination serology in the absence of MS isolation. In all cases, HI serology for Mycoplasma gallisepticum (MG) and M. meleagridis was negative. Acholeplasma laidlawii was isolated from some hens in each of these MS-seropositive culture-negative flocks. Immunoblotting was used to help determine if this positive MS serology was a result of cross-reactive antibodies to A. laidlawii or to some other Mycoplasma species. When sera from two of the flocks were reacted with MS antigen in immunoblotting, a strong and characteristic MS immunoblot profile was seen. Immunoblotting gave no evidence of a strong antibody response to A. laidlawii, M. iowae, or MG. This suggests the presence (or earlier presence) of MS in these flocks that is difficult to isolate by routine methods. Furthermore, this work shows that immunoblotting can be an important tool in the diagnosis of poultry diseases.     

Identification of Mycoplasma gallisepticum by use of monoclonal antibody in a rapid slide agglutination test.

Monoclonal antibody (MAb) against Mycoplasma gallisepticum strain PG31 was produced in BALB/c mice. The MAb (designated M9) was of IgG3 isotype and reacted with an epitope in M gallisepticum antigens with molecular weights of 35, 90, 95, and 98 kilodaltons (kDa). The M9 reacted with M gallisepticum antigens in the dot-blot ELISA and in western blot assays. It agglutinated M gallisepticum strains PG31, F, R, S6, A5969, and 9 field isolates from various sources. A coagglutination assay, using Staphylococcus aureus (Cowan strain 1), was developed to enhance the agglutination of some weakly agglutinating M gallisepticum isolates. The M9 did not react with M synoviae, M iowae, M meleagridis, M gallinarum, or M gallinaceum in any of the aforementioned assays. This MAb may be useful in facilitating laboratory diagnosis of M gallisepticum infections.     

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

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

Hemagglutination-inhibition versus serum plate agglutination in detecting Mycoplasma gallisepticum in broiler flocks.

Sera from 43,040 broilers in 36 flocks were tested for antibodies to Mycoplasma gallisepticum by serum plate agglutination (SPA) and hemagglutination-inhibition (HI) tests. The importance of testing SPA-negative samples for HI antibodies was demonstrated.     

鹅败血支原体及滑液囊支原体感染的血清学调查

本研究对珠江三角洲部分地区鹅群的185例血清样品，进行败血支原体、滑液囊支原体感染的平板凝集试验，检测结果表明：鹅败血支原体的感染率为29.7%，滑液囊支原体的感染率为19.5%，两者的混合感染率为14.6%。     

Monoclonal antibodies that recognize specific antigens of Mycoplasma gallisepticum and M. synoviae

The polypeptide profiles of the type strains of Mycoplasma gallisepticum (PG 31) and M. synoviae (WVU 1853) resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were compared. Except for a few discrete peptides that were similar, the species varied considerably in peptide profiles. Congruence was observed between the type strains of each species and homologous cloned serotypes. Protein blots of each species were probed with 2 mouse monoclonal antibodies. Monoclonal antibody G 46 was specific for the antigen p 110 (G) in M. gallisepticum, and S 221 was specific for an antigen complex p 45-50 (S) in M. synoviae. The 2 monoclonal antibodies clearly distinguished between all serotypes of M. gallisepticum and M. synoviae that were examined by Western blot transfer. Autoradiographs of 125I-labeled M. gallisepticum and M. synoviae indicated that p 110 (G) and p 45-50 (S) were surface membrane peptides. Indirect immunofluorescence of M. gallisepticum and M. synoviae in Vero cell cultures supported the autoradiographic findings. The p 110 (G) antigen of M. gallisepticum was heat-stable, pronase-sensitive, and resistant to periodate oxidation, suggesting that its chemical composition is protein. In contrast, the p 45-50 antigen complex of M. synoviae appeared as a broad band in protein blots treated with monoclonal antibody S 221, was sensitive to pronase, and responded to Schiff's reagent but was not completely inhibited by periodate oxidation, suggesting that it is a complex of repeating sequences probably composed of glycosylated peptides.     

Health survey of backyard poultry and other avian species located within one mile of commercial California meat-turkey flocks.

A survey was conducted to characterize domestic and exotic bird populations, estimate seroprevalence to selected disease agents, and describe health management practices on 62 premises containing "backyard" flocks located within one mile of 22 commercial California meat-turkey flocks participating in National Animal Health Monitoring System (NAHMS). Chickens were present on 56 backyard premises and turkeys on seven. Antibodies were identified against Mycoplasma gallisepticum, M. synoviae, M. meleagridis, Salmonella pullorum, Newcastle disease virus, avian encephalomyelitis virus, Bordetella avium, hemorrhagic enteritis virus, infectious bronchitis virus, and infectious bursal disease virus in 367 blood samples from 32 backyard premises. Twenty-two owners of backyard premises said they restricted visitor contact with their birds, and two required visitors to wear rubber boots and use boot disinfectant. Owners of seven premises used biologics and/or pharmaceutics for disease prevention. One family member worked on a commercial turkey ranch, but no other contact between owners, relatives, or employees and commercial poultry was reported.