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Genotyping of Mycoplasma bovis isolates using multiple-locus variable-number tandem-repeat analysis.
Luís Pinho1, Gertrude Thompson, Ricardo Rosenbusch
1ICBAS, Department of Veterinary Clinics, Instituto de Ciências Biomédicas Abel Salazar, University of Porto, R. Padre Armando Quintas, 7, 4485-661 Vairão, Vila do Conde, Portugal.
Multiple-locus variable-number tandem-repeat analysis (MLVA) offers a reliable method for differentiating Mycoplasma bovis isolates. This technique provides a fast, cost-effective approach for tracking the epidemiology of M. bovis in livestock.
Area of Science:
- Veterinary Microbiology
- Molecular Epidemiology
- Bovine Infectious Diseases
Background:
- Mycoplasma bovis is a significant pathogen causing mastitis, pneumonia, and arthritis in cattle, leading to substantial economic losses.
- Previous epidemiological studies on M. bovis isolates have been hampered by heterogeneous results from various typing techniques, hindering inter-laboratory comparisons.
- A need exists for a reliable and comparable molecular typing method to analyze geographically and temporally distinct M. bovis isolates.
Purpose of the Study:
- To develop and evaluate a multiple-locus variable-number tandem-repeat analysis (MLVA) scheme for differentiating Mycoplasma bovis isolates.
- To assess the genetic diversity of M. bovis isolates using the developed MLVA technique.
- To compare the performance of MLVA with established methods like random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE).
Main Methods:
- Developed an MLVA typing scheme based on nine selected tandem-repeat sequences from the M. bovis PG45 genome.
- Analyzed the genetic diversity of 37 M. bovis isolates from diverse geographic and temporal origins.
- Compared MLVA results with RAPD and PFGE, evaluating cluster concordance using Adjusted Rand and Wallace coefficients at various similarity cutoff levels.
Main Results:
- MLVA demonstrated a high Simpson's index of diversity (0.91), comparable to RAPD (0.99) and PFGE (0.99).
- Acceptable cluster concordance (Wallace coefficient ≥0.5) was achieved with MLVA at a relaxed cutoff of 52%, compared to 29% for PFGE and 18% for RAPD.
- The MLVA assay is compatible with standard PCR and gel electrophoresis, as well as high-throughput automated methods.
Conclusions:
- The developed MLVA scheme is a fast, reliable, and cost-effective method for differentiating M. bovis isolates.
- This MLVA technique provides a valuable tool for the surveillance of M. bovis epidemiology in the livestock industry.
- MLVA offers a standardized and comparable approach for molecular typing of M. bovis, overcoming limitations of previous methods.
