S. H. Kleven

Airsacculitis in broilers from Mycoplasma synoviae: effect on air-sac lesions of vaccinating with infectious bronchitis and Newcastle virus

SUMMARY Broilers vaccinated against infectious bronchitis and Newcastle disease developed airsacculitis following aerosol exposure to broth cultures of Mycoplasma synoviae. Incidence and severity of lesions was maximum 3 weeks postexposure, but few lesions were grossly visible 6 weeks postexposure. In another trial, aerosol exposure of unvaccinated birds or bronchitis vaccination 5 days after mycoplasma exposure resulted in a low incidence of airsacculitis with mild lesions. Vaccination 5 days prior to mycoplasma exposure led to increased severity of airsacculitis, while vaccination and mycoplasma exposure on the same day resulted in the highest incidence and severity of lesions. Aerosol exposure to M. synoviae in broilers vaccinated simultaneously against both infectious bronchitis and Newcastle disease led to depressed growth and less efficient feed conversion.

Pathogenicity of two strains of Mycoplasma gallisepticum in broilers.

Strains F and R of Mycoplasma gallisepticum (MG) were compared in two laboratory trials for their relative pathogenicity in terms of inducing airsacculitis and antibody production to MG. Chickens exposed to the R strain had significantly higher incidence of air-sac lesions (P less than 0.05) and greater severity of airsacculitis than did chicks exposed to the F strain. In both trials, chickens vaccinated simultaneously with Newcastle disease-infectious bronchitis vaccine and exposed to MG had more severe lesions than did chickens exposed to mycoplasma alone. chickens exposed to the F strain had significantly lower geometric mean hemagglutination-inhibition antibody titers to MG than did chicks exposed to the R strain. Chickens vaccinated simultaneously with Newcastle disease-infectious bronchitis vaccine and exposed to R strain had significantly lower body weights than did chickens in the other group.

Evaluation and Comparison of Various PCR Methods for Detection of Mycoplasma gallisepticum Infection in Chickens

Abstract Four generic Mycoplasma gallisepticum (MG) polymerase chain reactions (PCRs) (16S rRNA PCR, three newly developed PCR methods that target surface protein genes [mgc2, LP (nested) and gapA (nested)]) were compared for analytical specificity and sensitivity and for diagnostic sensitivity (Se) and specificity of detection from tracheal swabs. The licensed MG DNA Test Kit Flock Chek test (IDEXX, Laboratories, Inc., Westbrook, ME) was as well evaluated for the diagnostic specificity and sensitivity of detection from tracheal swabs. Analytical specificity was evaluated for the four generic PCR methods using a panel of DNA samples from microorganisms that may be isolated from the trachea of commercial poultry and other fowl. PCR methods mgc2, nLP, and ngapA only amplified DNA from MG, whereas 16S rRNA PCR amplified DNA from MG and Mycoplasma imitans. The analytical sensitivity of the four generic PCR methods expressed in color-changing units (CCU)/amplification reaction was estimated for each PCR method and ranged from 4 to 400 CCU/reaction; the sensitivities of single PCR methods 16S rRNA and mgc2 were estimated at 40 CCU/reaction, the nLP at 400 CCU/reaction, and the ngapA at 4 CCU/reaction. The diagnostic sensitivity and specificity of MG detection from tracheal swab pools, as compared to isolation from choanal cleft swabs, was evaluated for the five PCR methods using three groups of birds exposed to vaccine strains ts-11 and 6/85 and to challenge strain R. All PCR methods were able to detect the vaccine strains and the challenge strain R directly from tracheal swabs, indicating that PCR primers from the different methods amplified divergent MG strains. Isolation and PCR results correlated satisfactorily among the three experimentally infected groups, with agreement values (k) ranging from 0.52 to 1.00. The ngapA, IDEXX, and mgc2 PCRs showed the best sensitivity (Se) ratios for detection of M. gallisepticum strains as compared to isolation. Compared to the ngapA and IDEXX PCR methods, the mgc2 PCR has a faster turnaround time, since this test consists of a single amplification reaction and the amplification product is detected by gel electrophoresis. Therefore, among the PCR methods evaluated in this study, the mgc2 PCR is the method of choice to further validate in the field.

Comparison of in vivo and in vitro methods for pathogenicity evaluation for mycoplasma gallisepticum in respiratory infection

This study was designated to examine the pathogenicity of several strains of Mycoplasma gallisepticum (R, F, S-6, 227 and A5969) and laboratory derived substrains. Preliminary results indicated that the nine M. gallisepticum strains differed markedly in their pathogenicity for chickens. A comparison was made between various in vivo and in vitro methods for quantitative evaluation of pathogenicity. Reproducibility, convenience, and relevance to clinical observations were considered. Two in vivo tests were employed. In one case 2-week-old chickens were infected with M. gallisepticum by aerosol. Air sac lesion score, ability to reisolate M. gallisepticum from trachea and air sac, and serological response to M. gallisepticum were determined 2 weeks post infection. The second test was based on the ability to reisolate M. gallisepticum 3 days after intratracheal inoculation at different dose levels. In this way it was possible to calculate a median tracheal infection dose for each of the strains tested, a parameter which reflected their ability to colonise this organ. Scanning electron microscopy (SEM) was used to examine changes in the surface morphology of the infected trachea and photometric analysis of the SEM lesion was performed. Certain strains multiplied profusely in the trachea of healthy birds without causing detectable pathological changes. Chick tracheal ring (TR) cultures were used for estimating pathogenicity in vitro. In the standardised TR system a method for quantitative evaluation of mycoplasma-TR interaction was devised. The use of the TR system as a model for evaluating in vitro pathogenicity was rapid and less subject to environmental variation than the in vivo tests. The test was economical with respect to time, space and birds.

Field Investigation of Mycoplasma gallisepticum Infections in House Finches (Carpodacus mexicanus) from Maryland and Georgia

A field study investigating the occurrence of Mycoplasma gallisepticum (MG) in house finches (Carpodacus mexicanus) was conducted in Maryland and Georgia. Eighty-eight finches were captured and examined grossly and microscopically for MG-related conjunctivitis. Serum samples were obtained for serum plate agglutination (SPA) and hemagglutination inhibition (HI) testing. Swabs from conjunctiva, sinus, and choanal cleft were inoculated into two mycoplasma broth media for culture and polymerase chain reaction (PCR) testing. From Maryland, 12 of 57 birds examined had gross conjunctival lesions. MG was isolated from 9 of the 12 affected birds and from three birds without gross lesions. Fourteen of 22 finches tested by PCR were positive for MG. Sixteen of 38 birds were positive for MG by SPA, and 9 of these had HI titers of 1:40 or 1:80. From Georgia, 3 of 31 finches examined had gross lesions; two of these were both culture and PCR positive for MG. Twelve birds were positive by SPA, and two of these had HI titers of 1:80. Histologic findings in birds with gross conjunctivitis from both locations were characterized by extensive epithelial and lymphoid hyperplasia as well as lymphoplasmacytic inflammation in conjunctival tissues; keratitis was rarely present. The source of MG infection in house finches is unknown, and further research is warranted to determine the prevalence and impact of this newly described disease.

Mycoplasma gallisepticum vaccination: effects on egg transmission and egg production.

The effects of Mycoplasma gallisepticum (MG) vaccination on egg transmission of MG and egg production were evaluated. Leghorn hens vaccinated with live MG (strain F), with strain F plus MG bacterin, with one dose of MG bacterin, or with two doses of MG bacterin all transmitted MG through the egg at a significantly lower level than unvaccinated controls. Hens vaccinated with two doses of MG bacterin had the longest lag before detectable transmission of MG through the egg. All vaccinated groups were protected against the egg-production drop seen in unvaccinated hens challenged with virulent MG.

Molecular variability of the adhesin-encoding gene pvpA among Mycoplasma gallisepticum strains and its application in diagnosis

ABSTRACT Mycoplasma gallisepticum is an important pathogen of chickens and turkeys that causes considerable economic losses to the poultry industry worldwide. The reemergence of M. gallisepticum outbreaks among poultry, the increased use of live M. gallisepticum vaccines, and the detection ofM. gallisepticum in game and free-flying song birds has strengthened the need for molecular diagnostic and strain differentiation tests. Molecular techniques, including restriction fragment length polymorphism of genomic DNA (RFLP) and PCR-based random amplification of polymorphic DNA (RAPD), have already been utilized as powerful tools to detect intraspecies variation. However, certain intrinsic drawbacks constrain the application of these methods. The main goal of this study was to determine the feasibility of using anM.gallisepticum-specific gene encoding a phase-variable putative adhesin protein (PvpA) as the target for molecular typing. This was accomplished using a pvpAPCR-RFLP assay. Size variations among PCR products and nucleotide divergence of the C-terminus-encoding region of the pvpAgene were the basis for strain differentiation. This method can be used for rapid differentiation of vaccine strains from field isolates by amplification directly from clinical samples without the need for isolation by culture. Moreover, molecular epidemiology of M. gallisepticum outbreaks can be performed using RFLP and/or sequence analysis of the pvpA gene.

Ribosomal RNA gene probes to detect intraspecies heterogeneity in Mycoplasma gallisepticum and M. synoviae.

Intraspecies genotypic heterogeneity among strains of Mycoplasma gallisepticum and M. synoviae was tested using genomic fingerprints with a ribosomal RNA (rRNA) gene probe. The organisms DNA was digested by a restriction endonuclease, electrophoresed, transferred to a nitrocellulose sheet, and hybridized with 32P-labeled pMC5 plasmid carrying the highly conserved rRNA genes of M. capricolum. The resulting hybridization patterns indicated a degree of genotypic heterogeneity among M. gallisepticum strains more pronounced than among the M. synoviae strains tested. Most importantly, the live vaccine F strain of M. gallisepticum could be distinguished from virulent field isolates of this species, enabling the detection and identification of the F strain in areas in which vaccination with this strain has taken place. Genomic fingerprints with an rRNA gene probe can thus be added to the battery of tools useful in taxonomy at the intraspecies level and in epidemiology of mycoplasmosis in poultry.

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 Freys 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 tests importance in Mycoplasma diagnostic serology. The HI test can serve to confirm positive SPA results.

Influence of strain of Mycoplasma synoviae and route of infection on development of synovitis or airsacculitis in broilers.

Two strains of Mycoplasma synoviae (WVU 1853 and F10-2AS) were compared for their relative pathogenicity in terms of airsacculitis and synovitis. Both strains produced air-sac lesions after aerosol exposure of chickens vaccinated against Newcastle disease and infectious bronchitis; both produced synovitis when inoculated into the foot pad. The WVU 1853 strain was more likely to result in synovitis, whereas the F10-2AS strain was more apt to produce air-sac lesions.