Use of molecular diversity of Mycoplasma gallisepticum by gene-targeted sequencing (GTS) and random amplified polymorphic DNA (RAPD) analysis for epidemiological studies Naola M. Ferguson, 1 Diego Hepp, 2,3 Shulei Sun, 1 Nilo Ikuta, 2,3 Sharon Levisohn, 4 Stanley H. Kleven 1 and Maricarmen Garcı ´a 1 Correspondence Maricarmen Garcı ´a mcgarcia@uga.edu 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 Received 21 September 2004 Revised 3 January 2005 Accepted 17 February 2005 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. INTRODUCTION Mycoplasma gallisepticum is a major problem in the poul- try industry worldwide, causing chronic respiratory disease of chickens and turkeys. Control of M. gallisepticum has generally been based on eradication of the organism from breeder flocks and maintenance of mycoplasma-free status in the breeders and their progeny by implementation of biosecurity. The rapid and widespread expansion of poultry in restricted geographical areas and the consequent re- emergence of mycoplasma infection have necessitated a re- evaluation of the strategies utilized to control mycoplasma infections of poultry. In areas where complete eradication is difficult to attain, vaccination with live vaccines is utilized as an alternative control strategy (Whithear, 1996; Kleven, 1997). Consequently, with the increased use of vaccination, more powerful tools to trace the source of contamination and to differentiate vaccine strains from circulating field Abbreviations: CDS, coding DNA sequence; DR, direct repeat; GTS, gene-targeted sequencing; p, passage; RAPD, random amplified poly- morphic DNA. 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. 0002-7642 G 2005 SGM Printed in Great Britain 1883 Microbiology (2005), 151, 1883–1893 DOI 10.1099/mic.0.27642-0