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Published on: September 5, 2013
A multiplex PCR assay for molecular capsular serotyping of Mannheimia haemolytica serotypes 1, 2, and 6
Cassidy L Klima1, Rahat Zaheer1, Robert E Briggs2
1Agriculture and Agri-Food Canada Research Centre, Lethbridge, Canada.
Abstract:
Mannheimia haemolytica is an important respiratory pathogen of ruminants. Of the 12 capsular serovars identified, 1 and 6 are most frequently associated with disease in cattle, while 2 is largely a commensal. Comparative analysis of 24 M. haemolytica genomes was used to identify unique genes associated with capsular polysaccharide synthesis as amplification targets in a multiplex PCR assay to discriminate between serotype 1, 2, and 6 strains. The specificity of serotype specific gene targets was evaluated against 47 reference strains representing 12 known serovars of M. haemolytica and 101 field isolates identified through antisera agglutination as serotypes 1, 2, or 6. The results suggest this simple and cost-effective serotype specific PCR assay can be used as an alternative to agglutination based techniques to serotype the majority of M. haemolytica collected from bovines, thus averting the need to use animals and invest in expensive sera development for agglutination assays. In addition, the gene targets identified in this study can be used in silico to identify serotype 1, 2, and 6 strains in sequenced M. haemolytica isolates without the need for culture based analysis.
Insights
A new multiplex PCR assay can differentiate Mannheimia haemolytica serotypes 1, 2, and 6. This cost-effective method offers an alternative to traditional agglutination techniques for bovine respiratory disease diagnostics.
Area of Science:
- Veterinary Microbiology
- Bacterial Pathogenesis
- Molecular Diagnostics
Background:
- Mannheimia haemolytica is a significant respiratory pathogen in ruminants.
- Capsular serovars 1 and 6 are commonly linked to bovine disease, while serovar 2 is often commensal.
- Accurate serotyping is crucial for understanding disease dynamics and developing control strategies.
Purpose of the Study:
- To develop a molecular diagnostic tool for rapid and specific identification of M. haemolytica serotypes 1, 2, and 6.
- To identify unique genetic targets for a multiplex PCR assay.
- To provide an alternative to traditional serotyping methods.
Main Methods:
- Comparative genomic analysis of 24 M. haemolytica isolates.
- Identification of unique genes involved in capsular polysaccharide synthesis.
- Development and validation of a multiplex PCR assay using reference and field strains.
Main Results:
- Unique gene targets were identified for serotype-specific amplification.
- The multiplex PCR assay demonstrated high specificity in discriminating between serotypes 1, 2, and 6.
- The assay successfully serotyped M. haemolytica isolates from cattle.
Conclusions:
- A simple, cost-effective multiplex PCR assay can reliably serotype M. haemolytica strains.
- This molecular method serves as a viable alternative to animal-based agglutination assays.
- Identified gene targets enable in silico serotyping of M. haemolytica from genomic data.
