Development of multilocus sequence typing (MLST) assay for Mycoplasma iowae

• We developed a multilocus sequencing typing (MLST) method for Mycoplasma iowae.
• MLST enabled strain differentiation directly from DNA without MI isolation.
• Six-loci full MLST differentiated 47 MI samples into 23 unique sequence types.
• Four-loci mini MLST differentiated the same 47 samples in to 20 sequence types.

Mycoplasma iowae (MI) infection is an economically and commercially important disease of turkeys. There are no sequence typing assays available for MI strain identification, the only available molecular tools for this purpose, are DNA fingerprinting assays. In addition to their low reproducibility, fingerprinting assays require isolation of the microorganism in pure culture, which is difficult for avian mycoplasma. Therefore, we propose a multilocus sequence typing (MLST) assay as the first genotyping assay for identification of MI. Based on the two available MI genomes on GenBank, 26 loci of housekeeping genes were identified and studied in a diverse sample set. Finally, six genes were selected for the newly developed MLST assay. The final sequence analysis of the six loci (total of 5019 bp) (dppC, ulaA, valS, rpoC, leuS, kdpA) allowed the differentiation of 47 MI samples into 23 unique sequence types. Moreover, when only 4 loci were used to type the same set of samples, they resulted in 20 unique sequence types. Analysis of phylogenetic trees and clonal groups generated by MLST displayed a high degree of agreement with geographical and temporal information of the tested samples. MLST is a highly reproducible molecular epidemiology assay that can be used to identify positive clinical cases directly from DNA samples. Therefore, it provides a useful tool allowing for better identification, control and eradication efforts.