Whole-genome sequencing as a regulatory tool for quality control of Leptospira strains for vaccines and diagnostics

The maintenance of well-characterized Leptospira seed strains is critical for vaccine production and diagnostic applications. Conventional methods such as the cross-agglutinin absorption test (CAAT), multilocus variable-number tandem repeat analysis (MLVA), and multilocus sequence typing (MLST) have provided useful information but remain limited in resolution and are not routinely applicable in industrial settings. In this study, 25 Leptospira strains from vaccine manufacturers and diagnostic laboratories were characterized using MLVA and whole-genome sequencing (WGS). MLVA achieved partial typing (72 %), whereas MLST from WGS data resolved 88 % of the strains. MLVA was precise for confirming the identity of strains with an expected genetic profile but lacked the resolution to identify strains that had been misidentified. Complete classification (100 %) was only achieved by WGS through average nucleotide identity (ANI), MLST, and analysis of the lic12008 gene INDEL to differentiate the Copenhageni and Icterohaemorrhagiae serovars. Importantly, eight strains (32 %) showed discrepancies relative to company records, revealing misclassification and possible cross-contamination in industrial collections. These findings demonstrate the superior accuracy of WGS for Leptospira strain typing and highlight its value as a routine quality control tool. We propose that WGS be incorporated as a regulatory requirement for strain authentication in vaccine production and diagnostic applications, to ensure genetic purity, enhance biosafety, and improving the reliability of leptospirosis prevention and control.