Immunoreagents for development of a diagnostic assay specific for Rathayibacter toxicus

Rathayibacter toxicus is Gram-positive bacterium causing gummosis of forage grasses, and it produces a corynetoxin responsible for livestock poisonings in Australia. R. toxicus is a USDA APHIS-listed Select Agent, threatening seed and livestock production areas in the U.S. We therefore are developing diagnostic assays to prepare for the introduction of the pathogen into the U.S. Using proteomic data, we identified proteins expressed in R. toxicus but not in other Rathayibacter spp. Recombinant proteins corresponding to extracellular domains of candidate proteins were used to raise polyclonal and monoclonal antibodies. Antibodies were evaluated for sensitivity and specificity in immunoassays against a panel of Rathayibacter spp. and near-neighbours. Polyclonal and monoclonal antibodies exhibited high sensitivity and specificity for R. toxicus in assays with cultured bacteria. The highest-performing immunoreagents were identified for further testing with field samples collected in Australia and for eventual development of an immunoassay for bacterial detection in grass samples.