Protein Biomarker Identification for the Discrimination of Field Isolates From the Rev.1 Vaccine Strain by MALDI-TOF MS.

Rev.1 is a live attenuated vaccine strain that is widely used to control brucellosis in small ruminants. For successful surveillance and control programs, rapid identification and characterization of isolates and reliable differentiation of vaccinated and naturally infected animals are essential prerequisites. Although MALDI-TOF MS is increasingly applied in clinical microbiology laboratories for the diagnosis of brucellosis, species or even strain differentiation by this method remains a challenge. To detect biomarkers, which enable to distinguish the Rev.1 vaccine strain from field isolates, we initially searched for unique marker proteins by comparison of the Rev.1 and 16M proteomes. We found 113 protein sequences of 16M that revealed a homologous sequence in the Rev.1 annotation and 17 of these sequences yielded potential biomarker pairs. MALDI-TOF MS spectra of 18 Rev.1 vaccine and 183 Israeli field isolates were subsequently analyzed to validate the identified marker candidates. This approach detected two genus-wide unique biomarkers with properties most similar to the ribosomal proteins L24 and S12. These two proteins clearly discriminated Rev.1 from the closely related 16M and the Israeli field isolates. In addition, we verified their discriminatory power using a set of strains from various origins and of different MLVA types. Based on our results, we propose MALDI-TOF MS profiling as a rapid, cost-effective alternative to the traditional, time-consuming approach to differentiate certain isolates on strain level.