: MALDI-TOF MS and Whole Genome Sequencing Confirm That Serotypes K, L and M Deviate from Well-Known Reference Strains and Numerous Field Isolates.

is one of the most important bacterial agents of respiratory diseases in poultry. For correct identification and characterization of this fastidious bacterium, reliable diagnostic tools are essential. Still, phenotypic tests are used to identify and serotyping is the most common method for characterization, despite known drawbacks and disadvantages such as divergent results, cross-reactivity between strains, or the non-typeability of strains. The intention of the present study was to evaluate MALDI-TOF MS and whole genome sequencing for the identification and characterization of . For this purpose, a selection of 59 well-defined reference strains and 47 field strains derived from outbreaks on Austrian turkey farms were investigated by MALDI-TOF MS. The field strains originated from different geographical areas in Austria with some of the isolates derived from multiple outbreaks on farms within a year, or recurrent outbreaks over several years. MALDI-TOF MS proved a suitable method for identification of to genus or species level except for 3 strains representing serotypes M, K and F. Phylogenetic analysis showed that most strains grouped within one cluster even though they were comprised of different serotypes, while serotypes F, K, and M clearly formed a different cluster. All field isolates from turkey farms clustered together, independent of the origin of the isolates, e.g., geographical area, multiple outbreaks within a year or recurrent outbreaks over several years. Whole genome sequencing of serotype M, K and F strains confirmed the extraordinary status and deviation from known fully-sequenced strains due to a lack of sequence similarity. This was further confirmed by alignments of single genes (16S-RNA and rpoB) and multilocus sequence typing although the demarcation was less obvious. Altogether, the results indicate that these three serotypes belong to a different species than , and might even be members of multiple new species.