Polymerase chain reaction-based discrimination of viable from non-viable Mycoplasma gallisepticum.

The present study was based on the reverse transcription polymerase chain reaction (RT-PCR) of the 16S ribosomal nucleic acid (rRNA) of Mycoplasma for detection of viable Mycoplasma gallisepticum. To determine the stability of M. gallisepticum 16S rRNA in vitro, three inactivation methods were used and the suspensions were stored at different temperatures. The 16S rRNA of M. gallisepticum was detected up to approximately 20-25 h at 37 °C, 22-25 h at 16 °C, and 23-27 h at 4 °C. The test, therefore, could detect viable or recently dead M. gallisepticum (< 20 h). The RT-PCR method was applied during an in vivo study of drug efficacy under experimental conditions, where commercial broiler-breeder eggs were inoculated with M. gallisepticum into the yolk. Hatched chicks that had been inoculated in ovo were treated with Macrolide 1. The method was then applied in a flock of day 0 chicks with naturally acquired vertical transmission of M. gallisepticum, treated with Macrolide 2. Swabs of the respiratory tract were obtained for PCR and RT-PCR evaluations to determine the viability of M. gallisepticum. This study proved that the combination of both PCR and RT-PCR enables detection and differentiation of viable from non-viable M. gallisepticum.

The development of diagnostic real-time TaqMan PCRs for the four pathogenic avian mycoplasmas.

Four avian mycoplasmas are commonly recognized as poultry pathogens: Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), Mycoplasma meleagridis (MM), and Mycoplasma iowae (MI). The avian mycoplasmas are associated with respiratory disease, synovitis and arthritis, poor performance, skeletal deformities, and embryo mortality. Three main approaches are used for the diagnosis of avian mycoplasmosis: isolation and identification, detection of antibodies, and molecular detection of the organism's nucleic acid by PCR. In recent years real-time PCR technology has revolutionized the way clinical microbiology laboratories diagnose infectious diseases, but so far only a limited number of diagnostic real-time PCRs have been proposed for avian mycoplasma diagnostics. We developed a complete set of reliable diagnostic real-time TaqMan PCR assays for the four pathogenic avian mycoplasmas. The selected genomic targets of the developed assays were species specific and intraspecifically conserved and included the 16S-23S intergenic spacer region of MS and MM, the upstream region to the 16S ribosomal DNA of MI, and highly conserved foci of the mgc2 gene of MG. The four assays were demonstrated to be highly specific and sensitive to their target avian mycoplasma, with detection limits of one copy per reaction mix for the MG assay and 10 copies per reaction mix for the MS, MM, and MI assays. We believe that the incorporation of the developed assays in avian mycoplasma diagnostics will contribute to the accuracy, efficiency, and feasibility of diagnosis of these pathogens.

Strain differentiating real-time PCR for Mycoplasma gallisepticum live vaccine evaluation studies.

Mycoplasma gallisepticum causes respiratory disease and production losses in poultry. Vaccination of poultry with M. gallisepticum live vaccines is an approach to reduce susceptibility to infection and to prevent the economic losses. The development and evaluation of live vaccines usually requires the involvement of several vaccine and challenge strains in the same experimental setup. Our goal was to develop a tool to allow the differentiation between a set of known M. gallisepticum strains in a quantitative manner. We developed 5 real-time PCR assays that absolutely differentiated between one of the five commercial and laboratory vaccine strains: F, ts-11, 6/85, K5831, K5054, and the challenge strain R low when tested on in vitro cultures. The assay K5831 vs. R low was also tested on specimens from live birds that were vaccinated with K5831 and challenged with R low, and successfully differentiated between the vaccine and the challenge strains in a quantitative manner. This preliminary in vivo application of the method also shed light on possible protection mechanisms for the M. gallisepticum K5831 vaccine strain.

Sialidase activity in Mycoplasma synoviae.

Eleven strains of the avian pathogen Mycoplasma synoviae were evaluated for the presence of sialidase activity with the use of the fluorogenic substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and the sialidase inhibitor 2-deoxy-2,3- didehydro-N-acetylneuraminic acid. The kinetics of in vitro growth in modified Frey medium were also assessed for each strain. Five strains had been isolated from clinically symptomatic chickens, and strains WVU 1853T and K3344 have been demonstrated to be capable of reproducing disease in specific-pathogen-free chickens. All strains exhibited sialidase activity, although the amount varied 65-fold among strains (P < 0.0001) from 1.3 x 10(-7) to 2.0 x 10(-9) activity units per colony-forming unit. Strains originally isolated from clinically symptomatic birds had more (P < 0.05) sialidase activity than strains from asymptomatic birds. Strain WVU1853T exhibited the most sialidase activity (P < 0.0001) and grew to the highest culture density (P < 0.0001) among strains, but across strains, the rank correlation of growth rate with sialidase activity was not significant. Negligible activity was detected in conditioned culture supernatant fluid. This is the first report of sialidase activity in pathogenic strains of M. synoviae, which suggests a potential enzymatic basis for virulence of the organism.

Use of molecular diversity of Mycoplasma gallisepticum by gene-targeted sequencing (GTS) and random amplified polymorphic DNA (RAPD) analysis for epidemiological studies.

A total of 67 Mycoplasma gallisepticum field isolates from the USA, Israel and Australia, and 10 reference strains, were characterized by gene-targeted sequencing (GTS) analysis of portions of the putative cytadhesin pvpA gene, the cytadhesin gapA gene, the cytadhesin mgc2 gene, and an uncharacterized hypothetical surface lipoprotein-encoding gene designated genome coding DNA sequence (CDS) MGA_0319. The regions of the surface-protein-encoding genes targeted in this analysis were found to be stable within a strain, after sequencing different in vitro passages of M. gallisepticum reference strains. Gene sequences were first analysed on the basis of gene size polymorphism. The pvpA and mgc2 genes are characterized by the presence of different nucleotide insertions/deletions. However, differentiation of isolates based solely on pvpA/mgc2 PCR size polymorphism was not found to be a reliable method to differentiate among M. gallisepticum isolates. On the other hand, GTS analysis based on the nucleotide sequence identities of individual and multiple genes correlated with epidemiologically linked isolates and with random amplified polymorphic DNA (RAPD) analysis. GTS analysis of individual genes, gapA, MGA_0319, mgc2 and pvpA, identified 17, 16, 20 and 22 sequence types, respectively. GTS analysis using multiple gene sequences mgc2/pvpa and gapA/MGA_0319/mgc2/pvpA identified 38 and 40 sequence types, respectively. GTS of multiple surface-protein-encoding genes showed better discriminatory power than RAPD analysis, which identified 36 pattern types from the same panel of M. gallisepticum strains. These results are believed to provide the first evidence that typing of M. gallisepticum isolates by GTS analysis of surface-protein genes is a sensitive and reproducible typing method and will allow rapid global comparisons between laboratories.

Differentiation of Mycoplasma gallisepticum strains using amplified fragment length polymorphism and other DNA-based typing methods.

Amplified fragment length polymorphism (AFLP) was used to type 34 strains of Mycoplasma gallisepticum (MG) including vaccine strains ts-11, 6/85, and F. Using AFLP, a total of 10 groups, with 30 distinguishable AFLP typing profiles, were generated in the analysis. The AFLP method was able to identify and differentiate both MG field strains from recent outbreaks and those that were epidemiologically related. The AFLP procedure will provide assistance in identifying the sources of mycoplasma infections. Vaccine strains were also differentiated from other field strains, which will be useful in the evaluation of vaccination programs. The AFLP discrimination potential was compared to other molecular typing techniques such as gene-targeted typing by DNA sequence analysis of the MG cytadhesin-like protein encoding gene, mgc2, and random amplified polymorphic DNA assay on the same MG isolates. The three assays correlated well with one another, with AFLP analysis having a much higher discriminatory power and reproducibility.

Specific detection and typing of Mycoplasma synoviae strains in poultry with PCR and DNA sequence analysis targeting the hemagglutinin encoding gene vlhA.

Mycoplasma synoviae is a major pathogen of chickens and turkeys, causing economic losses to the poultry industry worldwide. In this study, we validated and applied polymerase chain reaction (PCR) and DNA sequence analysis on the N-terminal end of the hemagglutinin encoding gene vlhA as an alternative for the detection and initial typing of field strains of M. synoviae in commercial poultry. PCR primers were tested against isolates of M. synoviae from various sources along with other avian mycoplasma and other bacterial species. The vlhA gene-targeted PCR assay was highly specific in the identification of M. synoviae, with a detection limit of 4.7 x 10(2) color changing units/ml. DNA sequence analysis of amplified products was also conducted to validate the potential for typing M. synoviae strains using the N-terminal region of the vlhA gene. To evaluate the test, we applied the PCR assay to tracheal swabs collected from chickens challenged with M. synoviae strain K1968 and compared the results to the serologic detection. The PCR assay was also evaluated directly on tracheal samples collected from commercial layers. Overall, this vlhA gene-targeted PCR is a useful tool for detection and initial typing of M. synoviae and can be applied in the preliminary identification of M. synoviae isolates directly from clinical samples.

Inactivation, storage, and PCR detection of Mycoplasma on FTA filter paper.

We evaluated the feasibility of using Flinders Technology Associates (FTA) filter paper for the inactivation and storage of mycoplasma DNA templates and their detection by the polymerase chain reaction (PCR). FTA paper is a cotton-based cellulose membrane containing lyophilized chemicals that lyses most types of bacteria and viruses. Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) cultures were spotted at various volumes on the filter paper and stored at different temperatures for various periods of time before performing PCR. MG and MS were readily detected at all time frames (1-60 days) independent of the volume applied (1-100 microl) or storage temperature (4 C-41 C). Sensitivity and specificity of the FTA-PCR were comparable to the standard diagnostic PCR, allowing the detection of MG/MS in field samples without interference by nontargeted mycoplasma. Analysis of 193 field samples by both methods showed nearly 100% agreement with serology and culture results. The long-term DNA stability at a wide range of temperatures makes the FTA cards a good alternative for collecting and simultaneously inactivating mycoplasma. It also offers the convenience of storage and transport of DNA in a cost-effective manner for further molecular analysis, such as restriction enzyme length polymorphism and nucleotide sequencing.