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The genotyping potential of the Mycoplasma synoviae vlhA gene
Mohamed M El-Gazzar1, Amy N Wetzel, Ziv Raviv
1Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA.
Abstract:
Mycoplasma synoviae (MS) continues to cause significant losses to poultry producers, and studying the epizootiology of infection is an important component of MS control. The partial variable lipoprotein hemagglutinin A (vlhA) gene is the only genomic target identified so far for MS sequence typing. The vlhA gene codes for two variable cell surface proteins, lipoprotein and hemagglutinin, and the proposed mechanism for the variation is gene conversion between a single expressed gene and an array of pseudogenes. The upstream portion of the vlhA gene is present in the genome in a single copy (not present in the pseudogenes), and it is the only part of the gene that can be used for targeted sequence typing. However, the 3' end of the vlhA single copy" as well as this region's discriminatory potential for genotyping purposes has not been established. The purpose of this study was to identify the exact limit and the genotyping potential of the vlhA single copy region. New PCR assays were developed to amplify the entire conserved region and part of the variable region of the vlhA gene. Amplification and sequencing were performed on a variety of MS samples and on in vitro sequential generations of a standard MS strain. Sequence analyses determined the site and composition of the most proximal sequence variation that could be attributed to a gene conversion event, and they predicted the end point of the vlhA single copy region. The results indicated that a currently available "revised Hammond" PCR spans the whole single copy of the vlhA gene and exploits the full genotyping potential of this MS genomic target. In addition, this study allows interesting insight into the gene conversion mechanism of MS and offers the opportunity for further investigation this mechanism in mycoplasmas.
Insights
This study clarifies the Mycoplasma synoviae (MS) vlhA gene's single copy region, confirming its utility for precise MS genotyping and control strategies in poultry production.
Area of Science:
- Veterinary Microbiology
- Poultry Health
- Molecular Epidemiology
Background:
- Mycoplasma synoviae (MS) causes significant economic losses in poultry.
- The variable lipoprotein hemagglutinin A (vlhA) gene is the sole genomic target for MS sequence typing.
- Understanding MS epizootiology is crucial for effective control.
Purpose of the Study:
- To define the precise boundaries of the single-copy upstream region of the MS vlhA gene.
- To evaluate the genotyping potential of this specific vlhA region.
- To investigate the gene conversion mechanism in MS.
Main Methods:
- Development of novel PCR assays to amplify conserved and variable portions of the vlhA gene.
- Amplification and sequencing of MS samples and in vitro generated strains.
- Sequence analysis to identify gene conversion sites and determine the vlhA single-copy region's endpoint.
Main Results:
- The study precisely mapped the endpoint of the vlhA single-copy region.
- Established that the "revised Hammond" PCR assay covers the entire single-copy region.
- Demonstrated that this region offers full genotyping potential for MS.
Conclusions:
- The "revised Hammond" PCR is effective for comprehensive MS genotyping using the vlhA gene.
- This research provides insights into MS gene conversion mechanisms.
- The findings support improved MS sequence typing and control strategies.
