The Mycoplasma gallisepticum 16S–23S rRNA Intergenic Spacer Region Sequence as a Novel Tool for Epizootiological Studies

Abstract

Mycoplasma gallisepticum (MG) contains two sets of rRNA genes (5S, 16S and 23S) in its genome, but only one of the two is organized in an operon cluster and contains a unique 660-nucleotide intergenic spacer region (IGSR) between the 16S and the 23S rRNA genes. We designed a polymerase chain reaction (PCR) for the specific amplification of the complete MG IGSR segment. The MG IGSR PCR was tested on 18 avian mollicute species and was confirmed as MG specific. The reaction sensitivity was demonstrated by comparing it to the well-established MG mgc2 PCR. The MG IGSR sequence was found to be highly variable (discrimination [D] index of 0.950) among a variety of MG laboratory strains, vaccine strains, and field isolates. The sequencing of the MG IGSR appears to be a valuable single-locus sequence typing (SLST) tool for MG isolate differentiation in diagnostic cases and epizootiological studies.

The development and application of aMycoplasma gallisepticumsequence database

“…The four genetic regions selected for analysis, with expected PCR product sizes and discrimination indices for targeted DNA sequencing, are presented in Table 1. Primer sequences, thermocycler programmes and PCR amplification methods used were as previously described (Ferguson et al, 2005;Raviv et al, 2007). Amplified DNA was purified using the BigDye † Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA), and submitted to the Georgia Genomics Facility (University of Georgia) for sequencing.…”

“…Despite the extraordinary point substitution rate of M. gallisepticum (Delaney et al, 2012), selected genomic targets have proven sequence stability (Ferguson et al, 2005;Raviv et al, 2007). Targeted genetic sequencing is also a highly reproducible strain differentiation method, allowing the development of a reference database and global comparisons between laboratories (Ferguson et al, 2005).…”

“…Targeted genetic sequencing is also a highly reproducible strain differentiation method, allowing the development of a reference database and global comparisons between laboratories (Ferguson et al, 2005). A significant advantage of targeted sequencing over RAPD is that is can be performed directly from clinical samples, without the requirement for mycoplasma isolation in pure culture (Ferguson et al, 2005;Raviv et al, 2007).…”

“…The next objective was to use this sequence data to characterize South African M. gallisepticum wild-type genotypes (hereafter referred to as ''wild-types''), determine their distribution within the country and their identity to M. gallisepticum vaccines and wild-types from other countries, and finally to assess the ability of the selected targets to differentiate South African wild-types. Four previously characterized genetic regions were selected for analysis: two genes encoding surface proteins involved in cytadhesion (M. gallisepticum cytadhesin 2 [mgc2] and gapA), one gene encoding a predicted conserved surface lipoprotein (MGA_0319), and the 16S-23S rRNA intergenic spacer region (IGSR) (Ferguson et al, 2005;Raviv et al, 2007). This research demonstrates the value of an M. gallisepticum sequence database, and is the first report on the diversity and distribution of M. gallisepticum wild-types in South Africa.…”

Development of a novel real-time polymerase chain reaction (PCR) assay by amplification of double target genes for quantitative detection of Mycoplasma gallisepticum

“…The 16S rRNA nucleotides are convenient for rapid sequence analysis and the presence of universal regions enables its amplification by PCR for subsequent analysis. Another important advantage of the 16S rRNA gene is that it is not prone to horizontal gene transfer (Raviv et al, 2007), and that polymorphism between copies of the gene in the chromosome are not common, due to recombination by gene conversion. Gene of fMG-2 was a conservative marker for detecting MG, some developed PCR approach based on this gene have higher specificity.…”