Vet. Res. 35 (2004) 199-212
DOI: 10.1051/vetres:2004006

Validation of the suppressive subtractive hybridization method in Mycoplasma agalactiae species by the comparison of a field strain with the type strain PG2

Marc S. Marenda^a, Edy M. Vilei^b, François Poumarat^c, Joachim Frey^b and Xavier Berthelot<sup>a

a</sup>  UMR INRA-ENVT 1225, école Nationale Vétérinaire de Toulouse, 23 chemin des Capelles, 31076 Toulouse, France
^b  Institute of Veterinary Bacteriology, University of Bern, Laengassstrasse 122, Postfach, 3001 Bern, Switzerland
^c  AFSSA Lyon, 31, avenue Tony Garnier, BP 7033, 69342 Lyon, France

(Received 17 March 2003; accepted 22 September 2003)

Abstract - The subtractive suppressive hybridization (SSH), a method that allows the identification of sequences that are present in one genome (tester) but not in the other (driver), is a promising technique for the comparison of Mycoplasma agalactiae pathogenic strains. The optimal conditions for SSH were established by subtracting the M. agalactiae type strain PG2 DNA from the M. agalactiae strain 5632 DNA. Because these two strains possess different vpma gene repertoires, 5632-specific vpma sequences (and possibly other 5632-specific sequences) were predicted to be retrieved by SSH. The subtracted tester DNA was PCR-amplified and cloned into the pGEM-T easy E. coli vector. Two independent libraries were generated and used to prepare individual probes that were tested by Southern blot with genomic DNA from various field isolates and mycoplasma reference strains. Sequence analysis of two overlapping clones showed that they potentially code for a large carboxyterminal portion of a new vpma ORF. Several DNA fragments homologous to insertion sequences were also found in 5632 and related strains. These preliminary data suggest that SSH is a powerful method to investigate differences between mycoplasma strains, and may be applied to molecular epidemiology, diagnostic, and host specificity or pathogenicity determinant discovery.

Key words: Mycoplasma agalactiae / subtractive hybridization / vpma / insertion sequence

Corresponding author: Marc S. Marenda m.marenda@envt.fr

© INRA, EDP Sciences 2004