Abstract
BackgroundIn this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. For this, an extraction protocol for Leptospira spp. was independently established in two separate laboratories. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was then evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. To verify discriminating peaks for the applied pathogenic strains, statistical analysis of the protein spectra was performed using the software tool ClinProTools. In addition, a dendrogram of the reference spectra was compared with phylogenetic trees of the 16S rRNA gene sequences and multi locus sequence typing (MLST) analysis.ResultsDefined and reproducible protein spectra using MALDI-TOF MS were obtained for all leptospiral strains. Evaluation of the newly-built reference spectra database allowed reproducible identification at the species level for the defined leptospiral strains and the field isolates. Statistical analysis of three pathogenic genomospecies revealed peak differences at the species level and for certain serovars analyzed in this study. Specific peak patterns were reproducibly detected for the serovars Tarassovi, Saxkoebing, Pomona, Copenhageni, Australis, Icterohaemorrhagiae and Grippotyphosa. Analysis of the dendrograms of the MLST data, the 16S rRNA sequencing, and the MALDI-TOF MS reference spectra showed comparable clustering.ConclusionsMALDI-TOF MS analysis is a fast and reliable method for species identification, although Leptospira organisms need to be produced in a time-consuming culture process. All leptospiral strains were identified, at least at the species level, using our described extraction protocol. Statistical analysis of the three genomospecies L. borgpetersenii, L. interrogans and L. kirschneri revealed distinctive, reproducible differentiating peaks for seven leptospiral strains which represent seven serovars. Results obtained by MALDI-TOF MS were confirmed by MLST and 16S rRNA gene sequencing.
Highlights
In this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods
Sample extraction procedure and MALDI-TOF MS measurements This study focused mainly on well-defined pathogenic leptospiral strains used for serodiagnostic purposes which belong to three genomospecies: L. interrogans, L. borgpetersenii and L. kirschneri
To assess the influence of the optional washing step in the sample preparation procedure for MALDI-TOF MS typing, regarding the quality of the protein spectra, we compared strains that were prepared with and without the optional additional washing step combined with the concentrator process
Summary
In this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. The causative agents are different serovars of pathogenic Leptospira strains, bacteria that belong to the order Spirochaetales. They can affect humans as well as a wide range of different mammals [1] while the clinical manifestations differ considerably [2,3]. In pigs [10,11] and cattle [12] still birth, abortion, and foetal birth deformities may occur. In horses Leptospira spp. play a role in the clinical manifestation of the Equine Recurrent Uveitis (ERU) [13]
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