Species-specific biotinylated DNA probe for the detection of Mycoplasma gallisepticum

A 5.5 kilobase DNA fragment from an Eco RI digest of the Mycoplasma gallisepticum genome was specific for the detection of M. gallisepticum. This 5.5 kb fragment was initially cloned into bacteriophage lambda gt11 followed by subcloning into the plasmid vector pGEM-3Z. The incorporation of a biotin label was accomplished by utilizing biotin-11-dUTP in a nick translation reaction. This probe, designated pMg6, reacts specifically with M. gallisepticum when tested against various mycoplasma DNAs in Southern blot hybridization analysis. Spot-blot hybridization data indicate the pMg6 is capable of detecting 800 pg of M. gallisepticum DNA.     

Cytadherence-deficient mutants of Mycoplasma gallisepticum generated by transposon mutagenesis

Cytadherence-related molecules of Mycoplasma gallisepticum strain R-low were identified by Tn4001 transposon mutagenesis with the hemadsorption (HA) assay as an indicator for cytadherence. Three Gm(r) HA-negative (HA(-)) colonies displaying a stable HA(-) phenotype through several successive generations in which gentamicin selection was maintained were isolated from four independent transformation experiments and characterized. Southern blot analysis showed that the transposon was inserted as a single copy within the genome of each of the HA(-) mutants, suggesting that the transposon insertion was directly responsible for their inability to attach to erythrocytes. Sequence analysis of the transposon insertion sites revealed that in two mutants, the transposon was inserted at two distinct sites within the gapA structural gene. In the third mutant, the insertion was mapped within the crmA gene, which is located immediately downstream of the gapA gene as part of the same operon. In vitro attachment experiments with the MRC-5 human lung fibroblast cell line showed that the cytadherence capabilities of the HA(-) mutants were less than 25% those of original strain R. Experimental infection of chickens, the natural host of M. gallisepticum, with each of the three mutants demonstrated significantly impaired colonization and host responses. These data demonstrate conclusively the role of both GapA and CrmA proteins in the adherence of M. gallisepticum to host cells in model systems and in vivo colonization. Furthermore, these results underscore the relevance of in vitro cytadherence model systems for studying the pathogenesis of natural infections in chickens.