Establishment and evaluation of standard strains of four major pathogenic Leptospira species in China

Objective To establish Chinese standard strains of four major pathogenic Leptospira species, i.e. L. interrogans, L. borgpetersenii, L. alexanderi and L. weilii, according to the Technical Requirements for Evaluation of National Standard Strains of Pathogenic Microorganisms (WS/T 812—2022).

Methods Four reference strains of Leptospira, i.e. 56601, 56604, 56615 and 56655, belonging to four pathogenic species of Leptospira were selected. The morphological characteristics of the 4 strains were observed under the dark-field microscope. Based on 16S rRNA gene sequencing, species identification of the 4 strains was performed. Multilocus sequence typing (MLST) was conducted, and the alleles and sequence types (STs) of the 4 strains were obtained. The morphological and genetic stability of the 4 strains among different generations was evaluated by using dark-field microscope, 16S rRNA gene sequencing and MLST analysis.

Results All the 4 strains had typical morphological characteristics of Leptospira. Species identification revealed that the 4 strains, i.e. 56601, 56604, 56615 and 56655, belonged to four pathogenic Leptospira species, L. interrogans, L. borgpetersenii, L. alexanderi and L. weilii, respectively. A total of four different STs were identified from the 4 pathogenic Leptospira strains. The morphological characteristics of the 4 strains were stable after successive generations. The stability experiments results showed that the sequences of 16S rRNA gene among different generations were 100.00% homologous. In addition, the ST types among different generations were completely consistent. The genetic stability of the 4 strains was good. The whole genome sequencing analysis revealed that the genome size was 3.93–4.69 Mb and the G+C content was 36.00%–40.87%.

Conclusion The 4 strains, i.e. 56601, 56604, 56615 and 56655, have typical morphological and biological characteristics of pathogenic Leptospira, and their molecular genetic characteristics were stable, indicating that they can be used as four standard strains of pathogenic Leptospira species in disease control, clinical diagnosis, teaching and scientific research and vaccine development in China.