The U.S. Select Agent Rathayibacter toxicus is a toxigenic gram-positive bacterium responsible for a lethal disease of livestock and wildlife in Australia. Annual livestock deaths in Australia due to consumption of contaminated ryegrass have raised concerns about the global spread of R. toxicus in ryegrass hay and seed.
We collected new isolates of R. toxicus from South Australia. The genetic structure of R. toxicus populations was determined to evaluate the efficacy of detection protocols and to understand the potential for emergence of new strains. Two genetic analyses were applied to isolates of R. toxicus collected in Australia from 1973 to 2014.
We are developing the in vitro (qPCR and endpoint PCR) and field deployable (LAMP: Loop-mediated Isothermal Amplification; and RPA: Recombinase Polymerase Amplification) diagnostics methods to specifically detected R. toxicus. Whole genome sequences (Illumina MiSeq) were generated for representative isolates from different geographic areas. The genome assemblies of different isolates were used to design target-specific primers and probes. A sensitive qPCR assay based on an rpoD region was developed to detect all R. toxicus isolates. A multiplex PCR based on genome-informed strategically identified genes was developed to discriminate among three genetically-distinct populations of R. toxicus. LAMP and RPA were developed to detect all R. toxicus populations in field set-up. Each amplification assay included a multi-target artificial internal control to enhance reliability and accuracy. The detection assays were validated in silico and in vitro; they accurately detected R. toxicus from infected annual ryegrass and successfully identified the R. toxicus population type. These sensitive detection methods are easy to implement and will be appropriate for routine diagnostics, pathogen and population specific surveys, disease management, and biosecurity decisions to support export/import of annual ryegrass.
Currently, we have sequenced the complete genomes of three populations of R. toxicus. The PacBio RS II and Illumina MiSeq sequencing platforms were used for genome sequencing. We are comparing the genomes for genomic variation among different clusters responsible for different functions. We are also analyzing the CRISPR-Cas system of R. toxicus to confirm the emergence of new population.
Rathayibacter genomics database
Complete genome sequence of Rathayibacter toxicus strain WAC3373 (Geographical location: Western Australia): Click here for LINK
Popset of gene sequences used for population genetics of Rathayibacter toxicus: Click here for LINK