Genotyping of Mycoplasma bovis isolates using multiple-locus variable-number tandem-repeat analysis

Mycoplasma bovis has been considered an important cause of mastitis, pneumonia, and arthritis in bovines with a highly detrimental economic impact in the livestock industry. Previous epidemiological studies, using different typing techniques showed that the isolates were usually heterogeneous and results were difficult to compare between different laboratories. Reliable and comparable molecular typing techniques using geographically and temporal distinct isolates have never been used. The objective of this study was to use multiple-locus variable-number tandem-repeat analysis (MLVA) for the differentiation of M. bovis isolates. This typing scheme was developed using the sequenced genome of the M. bovis PG45 type strain. Nine tandem-repeat sequences were selected and the genetic diversity of 37 isolates of wide geographic and temporal origins was analyzed. The results were compared to those obtained with random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) for the same isolates. Cluster concordance between techniques was evaluated using Adjusted Rand and Wallace coefficients, and different cutoff levels of similarity were tested. Acceptable values of ≥0.5 for all techniques for the Wallace coefficient were only observed at the most relaxed cutoff level, i.e., 52% for MLVA, 29% for PFGE and 18% for RAPD. The Simpson's index of diversity was 0.91 for MLVA, 0.99 for RAPD analysis and 0.99 for PFGE. This MLVA assay is compatible with simple PCR and agarose gel-based electrophoresis steps as well as with high-throughput automated methods. The molecular typing scheme presented in this study provides a fast, reliable, and cost-effective typing method for surveillance of M. bovis epidemiology.