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

¹ Department of Avian Medicine, Poultry Diagnostic and Research Center, The University of Georgia, Athens, GA 30602-4875, USA
² Universidade Luterana do Brasil, Canoas, Rio Grande do Sul, Brazil
³ Simbios Biotecnologia, Canoas, Rio Grande do Sul, Brazil
⁴ Division of Avian and Aquatic Diseases, Kimron Veterinary Institute, Beit Dagan 50250, Israel

Correspondence
Maricarmen García
mcgarcia{at}uga.edu

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.

The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this work are AY556071–AY556382.

Six dendrograms constructed for GTS analysis are available as supplementary data with the online version of this paper.